Companies with interesting biote

Companies with interesting biotechnology 2011 Biotechnology W4034/W3034

Wed Nov 16 13:18:03 2011 Lynch Jim [email protected]

Nanosphere

As the number of sequenced human genomes increases, correlations to specific genetic diseases are becoming more prevalent. However, the mere presence or absence of a specific gene sequence is not always indicative of the presence or absence of a particular protein which may be responsible for the disease. However, correlating an individual’s DNA to the presence or absence of a particular protein is currently a time consuming process involving a variety of diagnostic techniques and testing protocols. It would be desirable to be able to use a single human sample, in conjunction with a single apparatus to test for both the presence of a particular DNA sequence and the presence or absence of a particular protein. Nanosphere Inc. has developed such a platform. Nanosphere Inc. The company was founded by a pair of Northwestern scientists, Drs. Robert Letsinger and Chad Mirkin. The technology takes advantage of a patented nanosphere particle. The particle itself is about 15nm and is comprised wholly of gold. Using this particle as a basis, Nanosphere has developed a proprietary platform called the VerigeneTM process. The Verigene process is comprised of a testing cartridge and a detector. Cartridges are directly inserted into the detector, and depending upon the cartridge, provide any of a sequence of 300-500bp, the presence or absence of a particular protein, and even whether particular viruses are present. While these tests can be performed by a variety of current techniques, the advantage of the Verigene process is that the analysis takes about ninety minutes, and uses a single sample derived from a patient. The detector uses differing cartridges depending upon the test being performed. Consequently, the Verigene process represents a significant time and savings advantage over currently available technologies. The Verigene system uses a cartridge similar to a traditional microarray chip. Each spot on the chip has an oligonucleotide attached. The oligonucleotide is anywhere from 300-500 nts long. A human sample is deposited in the detector, where it is sonicated to form fragments of 300-500 nts. These fragments are allowed to hybridize to the cartridge. A washing step removes unbound DNA. Then the nanosphere is added via the detector system. The nanospheres are gold particles to which oligonucleotides are attached (300-500nt). These particles are allowed to hybridize to the hybridized DNA fragments. The result is that the DNA bound to the cartridge is sandwiched between the gold particle and the slide. A second wash step removed unbound nanospheres. Silver is then deposited on the nanosphere to increase the ability of the particle to scatter light. The signal generated by the spheres is so strong that they are visible to the naked eye. A simple colorimetric analysis is used by the detector, which correlates the light refraction to the position, and returns the proper DNA sequence from a database. Thus the system is akin to a barcode analysis via a third generation sequencing system. The whole process takes no more than 90 minutes. A similar system is used for the presence of proteins. Instead of using oligonucleotides, however, the Verigene system uses a series of antibodies specific for a protein of interest. The same detector is used to verify the presence or absence of a particular protein. Again, the process takes about 90 minutes. Overall, the Verigene system is a powerful diagnostic tool. The signal generated by the nanosphere is visible to the naked eye, so no PCR amplification step is required. This avoids potential replication errors and as a result the Verigene system can be used to accurately test for SNPs. It is also the reason that the tests can be performed in such a short amount of time. Accordingly, the Verigene system represents a cheap, fast and accurate way to perform a variety of Analyses. SOURCES 1. www.nanosphere.com 2. US Pat. No. 7,259,252 3. Dobson, Mark G., Galvin, Paul, and Barton, David E., Emerging Technologies for Point of Care Genetic Testing. Expert Rev. Mol. Diagn 7(4) 359-370 (2007).

Mon Nov 28 00:18:34 2011 Bole Raevti [email protected]

Synthetic Genomics

Biodiesel is a biofuel that is commonly used as an alternative to petrodiesel in diesel engines. It is renewably produced from crops such as corn, potato and sugarcane, and as a carbon-neutral fuel, it is extremely important for the stabilization of CO2 emissions. Using biodiesel in cars also reduces the emission of sulfur and other smog producing particles. However one of the major problems concerning its use is its method of production. Manufacturing biodiesel in large quantities necessitates the use of large tracts of arable land for fuel production, as opposed to for food. While the benefits of using biodiesel are many, the world food supply would be crippled if it was our main biofuel. Current research is focused on harnessing the power of photosynthetic organisms such as algae to produce hydrocarbon-like biofuels. This process can be slow because the algae require many processing steps to harvest the required lipids. However, Synthetic Genomics has created algae that can continuously secrete oil on an industrial level. The use of algae for biofuel rather than food crops is much more sustainable because they require much less space, and can grow on land that is unsuitable for crops or living. Wild type algae store the oil as a source of energy, but Synthetic Genomics managed to insert the secretion pathway from another organism into the surface membrane of the algae, resulting in algae that simply release the desired oil. Furthermore, more oil is produced by the genetically engineered strain. Synthetic Genome has also been successful in creating the first entirely synthetic bacterium, by using Mycoplasma gene sequences to create the desired genome and then using it to replace the original genome of the creature. This was the first experiment in which the organism actually survived despite having an artificial genome. The implications of this company’s work are enormous, and range from manufacturing medicines to biofuels using these synthetic microorganisms. Their technology may, in future, allow us to decrease healthcare costs and also to lessen the impact of climate change. Sun

Dec 04 22:15:49 2011 Wang Brian [email protected]

Cellectis

Cellectis Transcriptional Activator-Like Effector Nucleases (TALENs) Transcriptional Activator-Like Effector Nucleases or TALENs are essentially DNA scissors that bind to and cut at specific sequences of DNA. The complex is made up of two domains: the transcriptional activator-like effector (TALE) and nuclease domain. TALE is a class of naturally occurring protein produced by plant pathogenic bacteria. The DNA specificity of TALE is determined by a central domain of tandem 33-35 amino acid repeats. In addition, there is a pair of polymorphic amino acids at position 12 and 13 that further specifies the target. Each of the four most common polymorphic amino acid pairs corresponds to one of the four nucleotides. The main advantage TALE is the predictable nature of their DNA specificity. By modifying the amino acid sequences of the tandem repeat region of TALE, we can virtually customize TALE to bind to any DNA sequence. It is due to this predictability in specificity that there has been great interest in using TALE in DNA targeting. In particular, TALE can be fused with a nuclease domain (creating TALEN) which can be used to target and create double stranded breaks. Double stranded breaks are repaired in the cell via two mechanisms: non-homologous end joining and homologous recombination. Non-homologous end joining can result in insertions or deletions and thus is an excellent method of disrupting a gene. Homologous recombination can be utilized in gene replacement or insertion techniques. Previously, such genomic modification techniques used zinc finger nucleases or homing nucleases, both of which are limited by the difficulty of creating new DNA specificities. Unlike TALENs, new DNA specificities with zinc finger nucleases and homing nucleases require a selection-based approach, which is not only difficult but time consuming. Being unbound by this constraint, TALENs is a more efficient approach to targeting and modifying DNA. Moreover, studies have shown that the ability of TALENs to mutagenize a cell is either better than or on par with zinc finger nucleases, exemplifying their effectiveness as well (T. Cermak et al. Nucleic Acids Research 2011, 39(12), e82). The implications of this technology are very widespread, covering many potential applications of genome modifications. These applications include DNA targeting to study organism gene functions, introduction of genes into plants for resistance to pathogens, and treatment of human genetic disorders. Focusing more on the treatment of human genetic disorders, most current therapeutics and research efforts rely on viral gene therapy, which are accompanied by a number of side effects. Most notable, the dangers of viral gene therapy include immune reaction against the therapy and unwanted non-specific insertions of genes. The plasticity of the TALE domain not only gives TALENs a wide range of DNA specificities, but it also makes this technique potentially safer for human gene therapies.

Mon Dec 05 19:12:15 2011 Mou Yufei [email protected]

Molecular Staging

I will focus my paper on Rolling Circle Amplification Technology (RCAT) which was recently introduced by Molecular Staging, Inc. In addition, Molecular Staging Inc. had awarded three additional patents for RCAT. It is an advanced technology which can be applicable to simultaneous detection of molecules as a potential tool in detection and monitoring of diseases by flow cytometry and combination with protein microarrays to improve sensitivity in multiplex immunoassays on microspheres. RCAT uses a circular DNA template and a highly specific polymerase to manufacture a long single-strand DNA sequence which contains thousands copies of the circle and a plenty of brightly fluorescent labeled dyes binding sites. The stained DNA strand by fluorescent dyes can attach to the target molecule which you are interested in resulting in facilitated detection. “Technology (RCAT™) can significantly improve the diagnosis of cancer, infectious diseases, neuropsychiatric disorders, auto-immune disorders, and other major disease states.”(by Molecular Staging Inc.) RCAT can support the expanding use of complex marker panels for disease diagnosis or prognosis. Antibodies are usually used to characterize a plenty of cells based on their surface markers. However, cells can be best characterized only when their markers is stained by brightly dyes to be clearly different from unstained cells. RCAT has been successfully applied to the detection of cell surface antigens on peripheral blood mononuclear cells (PNMCs) such as CD4 and CD28 (by Davis KA et al.). The RCAT have achieved a more than 10 fold increase in median fluorescent intensity (MFI) compared with conventional detection methods ( Gusev Y et al.). “Rolling circle amplification technology (RCAT) can be applied to agglutination, enzymatic, and fluorescence detection systems covering all of the detection methods commonly used with beads. In each of these formats, RCAT offers significantly improved sensitivity.” (by Molecular Staging Inc.). RCAT is adaptable to an on-chip signal amplification. In RCAT, a circle of DNA, a short DNA primer (complementary to a portion of the circle) and an enzyme catalyst converts dNTPs into a single-stranded concatameric DNA molecule that is composed of thousands of tandemly repeated copies of the circle. Unlike other amplification procedures, RCAT produces a single amplified product that remains linked to the DNA primer. Consequently, RCAT is well suited to solid phase formats such as microarrays to generate localized signals at specific microarray locations. This distinctive property of RCAT should allow many assays to be performed simultaneously (multiplexing) without interference. In immunoRCA, the 5’end of an RCA primer is attached to an antibody. Thus, in the presence of circular DNA, DNA polymerase, and nucleotides, the rolling circle reaction produces a concatamer of the complement of the circular DNA sequence that extends from the end of the original primer remaining attached to the antibody. The amplified DNA can be detected by hybridization of complementary oligonucleotide probes or by antibodies specific for nucleotide analogs during the RCA reaction. Reference: [1]:Davis KA, Lin Y, Abrams B, Jayasena SD. Staining of cell surface human CD4 with 2’-F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic Acids Res 1998;26:3915–24. [2]:Gusev Y, Sparkowski J, Raghunathan A, Ferguson H Jr, Montano J, Bogdan N,et al. Rolling circle amplification: a new approach to increase sensitivity for immunohistochemistry and flow cytometry. Am J Pathol 2001; 159:63–9.

Mon Dec 05 19:12:26 2011 Mou Yufei [email protected]

Molecular Staging

Tue Dec 06 01:02:26 2011 Quinn Stuart [email protected]

Twist DX

The molecular biology epoch was ushered in largely due to advances in biotechnologies including the development of the polymerase chain reaction (PCR). Despite its enormous utility the PCR remains a biotechnology that is confined to scientific and medical diagnostic laboratories. The main barrier to PCR becoming truly a ubiquitous tool is the price of requisite thermocyclers, which cost several thousand dollars for simple machines and tens of thousands for those capable of quantitation. Twist DX has come up with a reaction that amplifies a single DNA molecule in an exponential manner with a fidelity approaching that of PCR in a shorter time. Moreover the Twist DX reaction occurs at a constant temperature therefore it does not require a thermocycler, which significantly reduces the cost. The reaction is based on a patented technology known as recombinase polymerase amplification (RPA). RPA chemistry is relatively simple; it makes use of a proprietary recombinase to anneal the primer to double stranded template without melting. Single-strand binding proteins stabilize the single-stranded region of template DNA long enough to allow polymerase to extend the molecule. This process then automatically repeats itself as the recombinase searches the double stranded DNA and inserts another primer to initiate another round of polymerization. As this process does not depend on cycling, the molecular machinery works at nearly maximal speed – never pausing for annealing or melting. This results in amplification from a single DNA template to detectable levels in approximately 5-10 minutes. Twist DX has taken its technology even further and generated a way to perform real time detection using fluorescent primers and quenchers. In this system, a tetrahydrofuran (THF) abasic site mimic is flanked by two nucleotide oligos. The upstream oligo is attached to the THF at its 3’ end, which also has a fluorophore attached. The downstream nucleotide element is linked to the THF at its 5’ end, which contains a quencher (also at the 5’ end) and a blocking group at its 3’ end. Once inserted by the recombinase complex, the down-stream nucleotide (containing the blocking group and quencher) is removed by an e. coli double-strand–speciﬁc endonuclease, Nfo, thus exposing a free 3’ OH group. In this way, extension and the fluorophore are activated simultaneously by Nfo and the fluorescence level can be monitored by a cheap, handheld fluorometer made by Twist DX. RPA is not revolutionary in its end but rather in its means: it is a cheaper, faster, and simpler way of amplifying DNA. The utility of such a reaction is that it will allow PCR-like reactions to reach out of the rarified environment of the scientific and diagnostic laboratory and infiltrate places such as high-school classrooms, restaurant kitchens, doctors’ offices in rural and impoverished regions and the basement laboratories of curious intellectuals. Such a move could be akin to the introduction of the personal computer wherein the public embraced a technology and its true potential was unleashed.

Tue Dec 06 12:50:13 2011 Hahn Philip [email protected]

Geron

Geron’s primary technological advances are focused on the activation and deactivation of telomerase, the enzyme that provides TTAGGG caps in vertebrates that protect DNA helixes and regulate the upper bound of cell division. The company intends to exploit their on/off control of telomerase for anti-cancer and HIV-related aging applications. A hallmark of cancer is the activation of telomerase, allowing colonies of cancer cells to reproduce without limit and leading to the death of the host organism. Geron has two candidates in clinical trials, the first being a Phase II trialed telomerase targeting GRN163L, which has shown efficacy against CD138+ and CD138- multiple myeloma cancer stem cells. GRNVAC1, a telomerase vaccine also in Phase II, is being tested with prostate cancer in the hopes of stimulating a greater immune response to the tumor. In a reverse tactic, Geron is conducting research on telomerase activation to delay cellular senescence. TAT0002 is their treatment candidate for HIV, and is a saponin clyoastragenol extract of the Astralagus family of shrubs. A spinoff licensed to TAsciences is TA-65, the active component of which is clyoastragenol. Among HIV’s deleterious effects is premature aging of tissues, potentially related to chronic inflammation and telomere shortening. Artificially increasing available telomerase could delay onset of this condition. Geron claims it can accomplish this feat without increasing oncogenic potential. Two early Geron researchers won the Nobel prize for their efforts in 2009. Biotech companies usually acquire or develop a second line of products, and Geron’s ‘other’ advances are in human embryonic stem cells. GRNCM1 is designed to remyelinate damaged axons, with applications in spinal cord injuries. Technologies used in the production of hESCs have been developed with Corning Life Sciences and Geron is able to produce hESCs in bulk. Through acquisition of Roslin Bio-Med, Geron retains the patent and technology licenses for nuclear transfer that arose from the pioneering cloning of Dolly the sheep. Safe nuclear transfer to enucleated cells has applications in animal cloning and agriculture, and Geron re-licenses the technology in a joint venture with Exeter subsidiary ViaGen. Geron’s combination of Phase II-progressed primary drug pipelines and potential profit generation through hESC production and cloning technologies render it on the shortlist of biotech investing. Its Phase III results if successful should provide a compelling investment case.

Tue Dec 06 17:44:30 2011 Dendy Meaghan [email protected]

Oxitec

Oxitec is a groundbreaking company that is utilizing biotechnology tools to make strains of sterile insects. They are employing this method to control insects such as mosquitoes that cause risks to both public health and local agriculture. The Sterile Insect Technique is the historically used method to control similar pests, but based on the use of radiation in that technique, mosquitoes are not a possible target since the radiation inhibits them from competing for mates in a natural environment. The Release of Insects carrying a Dominant Lethal genetic system (or RIDL as they refer to it) is a novel approach that shows an improved ability to sterilize mosquitoes and similarly difficult insect species while still allowing them to compete for possible mates. Their website also states that the RIDL method includes novel methods for separating sexes and for marking/monitoring the specimens. As of now, the company advertises Bisex RIDL strains, Aedes aegypti OX513A and pink bollworm OX3402, and Female-specific RIDL strains, Aedes aegypti OX3604C and Mediterranean fruit fly OX3647. The OX513A system, for example, uses a repressible lethal genetic system that allows the mosquitoes to grow to adulthood in the presence of tetracycline but their offspring will not be able to survive without the repressor tetracycline in its diet. There is a positive feedback system composed of a tetO binding domain, a minimal promoter, and the tTA gene which allows for the minimal promoter to make tTA which then binds to the tetO binding sites when tetracycline is absent. This tTA binding allows for the production of even more tTA which binds to even more tetO binding sites. When this has happened enough the high levels of tTA kill the cells. All of this is halted in the presence of tetracycline which is able to bind the tTA and block it from binding to the binding sites, which allows the cells to live. The effective monitoring system referenced by the company is also quite simple and very innovative. Oxitec utilizes an inherited fluorescent marker when creating their strains to allow for researchers to quickly identify offspring and sterilized males in the field while collecting data. This is improved when compared to the fluorescent dusts and food dyes used prior, which might not stay in place efficiently and cannot be inherited. I chose Oxitec based on how they’ve taken a simple biotechnology method in a Tetracycline system and turned it into a number of strains that can positively impact parts of the world that are plagued by diseases and agricultural issues caused by species such as Aedes aegypti. Their positive results in a recent experiment, in Grand Cayman, also shows how driven and forward-thinking this company is. In releasing just over 3 million genetically-altered male mosquitoes on Grand Cayman, the company saw about an 80% decrease in mosquito population, which is amazing when one considers the cases of dengue contracted in the area. This company and their innovative biotechnology method brings an answer to those insect carriers that the Sterile Insect Technique cannot effectively eradicate.

Tue Dec 06 19:29:15 2011 Baier Felix [email protected]

Optogenetics (LucCell)

We chose to write about optogenetics because it has recently revolutionized the entire field of neuroscience and may even extend its impact to other disciplines such as immunology or cardiology. Alongside, the technique has also attracted interest from the biotechnology industry, and first applications in clinical medicine are currently being tested. What is the revolutionary potential of optogenetics? Attributing specific function to single cells, circuits or entire regions in the brain is a real challenge for modern biology, that can only be approached with the help of tools to record and control activity of specific neurons in model organisms. Traditionally, the standard method for this purpose was electrophysiology: Electric currents could be injected in neurons to induce their firing, and action potentials decoded with intracellular electrodes. However, the method has serious flaws. Optogenetics not only avoids all the problems associated with electrophysiology, but offers additional benefits. So what then is optogenetics exactly? Optogenetics is essentially the optic control (“opto”) of neuronal activity via light-sensitive ion channels expressed (“genetics”) in target cells. In recent years, a large collection of light-sensitive ion channels with different function has been isolated from diverse organisms, such as bacteria and green algae. For example, ion channels might (passively) sense and report neuronal activity (=sensors), or might (actively) interfere with cell activity to change its activation pattern (=actuators; activation vs. inhibition). Importantly, unlike traditional electrophysiology, optogenetics is a non-invasive technique: delivery of a short light pulse is enough to trigger or inhibit the ion channel. Obviously, this makes it possible to control neuronal activity in the freely moving animal. Also, virtually any cell that has a distinct gene expression profile (either anatomically or functionally) can be precisely equipped with engineered ion channels. In traditional electrophysiology, targeting of sharply delimited pools of neurons, in contrast to individual neurons or entire tissue regions, was difficult or even impossible. In addition, optogenetics works in the millisecond dynamic range necessary to monitor and control neuronal activity. Taken together, these benefits will make it possible for the first time to address a major new field of investigation: probing the spike pattern of more than one cell or cell type during simultaneous activation, thereby mimicking the real-time action of entire neuronal circuits. While the field is still young, there have already been attempts to use optogenetics for the direct benefit of diseased people. The start-up company LucCell, for example, currently investigates the potential of optogenetics to restore function in paralyzed patients. At least in a model organism, their approach shows great promise: When nerve cells that control the diaphragm were activated with the optogenetic technology, mice that had been partially paralyzed showed resumed diaphragm muscle function. LucCell now studies bladder function, which is also affected by paralysis and could be restored by inhibiting the neurons that relax the bladder. Overall, therefore, optogenetics has great potential both for basic science and clinical applications, and clearly deserved to be elected “Method of the Year” by Nature Methods in 2010.

Tue Dec 06 21:08:41 2011 Kato Niyo [email protected]

Oxitec Technology:

RIDL (Release of Insects carrying a Dominant Lethal genetic system) Sterile Insect Technique (SIT) has been used since the 1950s as a species-specific, sustainable, and environmentally friendly method of pest control (Oxitec). It involves mass-releasing sterilized insects that reduce the wild population through infertile mating (Fu et al., 2007; Harris et al., 2011). Until recently, ionizing radiation was the sole method by which effectively sterilized insects could be obtained. However, irradiation has been found to be too damaging for many insect species, which is why the use of SIT has been limited to date (Fu et al., 2007). Oxitec’s RIDL technology uses modern biotechnology to circumvent this issue, making SIT a more efficient, economic, and purportedly safer than traditional method of pest control (chemical pesticides, swamp draining, or insect nets) (Ostera and Gostin, 2011; Oxitec). The fundamental principal of their products involves exploiting sex-specific alternative splicing and a Tet-Off positive feedback system to generate male insects that carry a female-specific autocidal system (Fu et al., 2007). One of their products is the Mediterranean fruit fly (Ceratitis capitata) in which, the gene “transformer” (Cctra) was used to construct the female-specific lethal system. Tra is spliced in female flies to produce three transcripts (F1, M1, and M2), only one of which produces a functional protein (F1), while males only produce the M1 and M2 transcripts, and therefore do not produce any Tra protein (Fu et al., 2007). Oxitec inserted the tra intron (spliced only in females), into a Tet-OFF positive feedback system, which has been previously shown to repressive dominant lethality in flies. In the absence of tetracyclin, tTA protein is produced, and enhances its own transcription in a positive feedback loop. This loop results in the build up of tTA over time, which in high concentration is lethal. By inserting the female-specific intron into this coding region, Oxitec was able to produce a female-specific lethal configuration, in which tTA is only made by female flies (Fu et al., 2007). The system is driven by an embryonic promoter to allow embryonic female-specific lethality, which is favorable for male-only releases (since female insects sometimes blood feed and damage crops through egg-laying) (Fu et al., 2007; Harris et al., 2011). The sex-specific lethal constructs also included a fluorescence Ds-Red gene to allow easy physical identification of transgenic insects (can also be confirmed via PCR) (Oxitec).This system is significantly better than prior methods of identification of transgenic insects, which used fluorescent dust, or food dye, neither of which is easily detectable and/or heritable (Fu et al., 2007). Oxitec’s RIDL technology is a clever system that exploits sex-specific alternative splicing to produce transgenic insects that are able mate competitively with their wild counterparts (Harris et al., 2011; Oxitec). In doing so, Oxitec has developed an safe and effective tool to fight disease and agriculturally deleterious pests on a global scale. References: Fu, G., Condon, K.C., Epton, M.J., Gong, P., Jin, L., Condon, G.C., Morrison, N.I., Dafa'alla, T.H., and Alphey, L. (2007). Female-specific insect lethality engineered using alternative splicing. Nat Biotechnol 25, 353-357. Harris, A.F., Nimmo, D., McKemey, A.R., Kelly, N., Scaife, S., Donnelly, C.A., Beech, C., Petrie, W.D., and Alphey, L. (2011). Field performance of engineered male mosquitoes. Nat Biotechnol 29, 1034-1037. Ostera, G.R., and Gostin, L.O. (2011). Biosafety concerns involving genetically modified mosquitoes to combat malaria and dengue in developing countries. Jama 305, 930-931. Oxitec. Our Research (Oxford Insect Technologies).

Tue Dec 06 21:37:40 2011 Khobrekar Noopur [email protected]

Biosearch Technologies

Biosearch Technologies: Stellaris™ FISH probes for in situ mRNA detection, quantification and visualization ‘Biosearch Technologies’ is a California-based Biotech company (founded in 1978) that focuses on designing and manufacturing innovative nucleic acid based products. Stellaris RNA FISH probes can be used to detect, localize and quantify mRNAs in situ in fixed cell samples using a simple, one-day procedure which doesn’t need isolation, purification, equalization or amplification of mRNA and hence shows no bias towards high copy number mRNAs (Orjalo, Johansson et al. 2011) The Stellaris FISH probes are 30-48 nucleotides long, labeled at the 3’end and have a high GC content. At least 30-50 probes hybridize to the same target mRNA which ensures high specificity of hybridization with target mRNA and amplifies the signal sufficiently to detect even a single mRNA molecule. Presence of multiple fluorescent probes in a single location in the cell is detected as a distinct diffraction-limited spot in a widefield fluorescence microscopy. The Stellaris probes are redundant oligonucleotides with uniform GC content which optimizes their binding to the target mRNA at a given hybridization stringency. Hybridization conditions of 30C and 10% (v/v) Formamide are generally used. For target mRNAs with more than 50-60% GC content, 25% (v/v) Formamide can be used for hybridization and washing. Very high stringency conditions often lead to high number of false positive results. Widefield microscopy limits the thickness of the samples that can be used for mRNA detection. Single mRNA signals are optimally detected when samples are 7-8 microns in thickness. The thicker samples lead to more out-of-focus light that obscures faint mRNA signals. (Raj and Tyagi 2010) Redundant oligonucleotides also ensure that only the mRNAs that bind multiple probes will show a signal that is read as above the threshold fluorescence, also as the probes are singly labeled, the stringency for detection increases. In other methods where we have heavily labeled probes, we can get a single probe that is bound non-specifically to the mRNA and still gives a signal that is above the threshold level. (Orjalo, Johansson et al. 2011) The mRNA quantification can be done for as low as 20 copies of mRNAs in cell in contrast to the semi-quantitative data produced by northern or western blot which gives relative concentrations of RNA and proteins in cells, respectively. A single spot in Stellaris FISH may represent conglomeration of multiple RNA molecules rather than a single RNA molecule bound to multiple probes. But it has been shown that only a single mRNA is present in one spot which can be confirmed using differentially labeled mRNAs. (Raj and Tyagi 2010) Applications: • Multiplex detection of multiple mRNAs simultaneously using various dyes is possible (only limited by the filters available for fluorescence microscopy) • Detects transcription site activity in the nucleus • Detects RNAs that are bound to interacting proteins or that are partially degraded (multiple probes still bind the target RNA to give detectable signal) References: Orjalo, A., H. E. Johansson, et al. (2011). "Stellaris[trade] fluorescence in situ hybridization (FISH) probes: a powerful tool for mRNA detection." Nat Meth 8(10). Raj, A. and S. Tyagi (2010). Chapter 17 - Detection of Individual Endogenous RNA Transcripts In Situ Using Multiple Singly Labeled Probes. Methods in Enzymology. G. W. Nils, Academic Press. Volume 472: 365-386.

Tue Dec 06 22:24:28 2011 Buchel Jason [email protected]

Synthetic Genomics

Synthetic Genomics is a company which makes synthetic microbial organisms in order to produce biofuels more efficiently. Improving this efficiency greatly improves many problems facing our environment. The company has previously produced strains of algae which excrete an oil used to refine the production of gasoline. Moreover, the cells successfully convert CO2 into hydrocarbons, which is very beneficial for producing biofuels. Using algae as opposed to other organisms for these purposes also has many advantages. For example, algae consume CO2, which could help to alleviate the Greenhouse Effect. In addition, algae can produce larger volumes of biofuels in comparison to using other sources. To produce these synthetic organisms, an artificial genome is first created then transplanted. The company originally synthesized an artificial genome of the Mycoplasma mycoides, and then transplanted this genome into its cousin, Mycoplasma capricolum. The newly created hybrid cells were able to incorporate the synthetic genome where only the donor mycoides proteins were being produced. To prove that this product was in fact synthetic, they were able to spell out in code email addresses and names of people leading the project. Yeast was used a means to produce the DNA, and watermarks were incorporated into the sequence in order to distinguish synthetic DNA from original. As an initial step to creating the synthetic genome, an antibiotic selection marker was incorporated into the mycoides genome. This allows selection of the transplanted chromosomes. Moreover, methylases were added to the genome to disable methylation protecting the cell during transplantation. They purified the DNA, then inserted it into the capricolum cells. Gel electrophoresis and sequencing were used to further verify that it was the donor mycoides proteins being expressed. The technology was coined genome transplantation. The company synthesized gene cassettes, which were a set of genes to be inserted into the synthetic genome. The cassettes were approximately 1,000 bp long. The cassettes contained both Asc I and BssH II restriction sites and were recombined with various vector elements to promote growth. They were recombined in yeast, then transferred to E.coli. They isolated plasmid DNA from clones and screened for cells with the insert. When it was not stable in E.coli, the DNA was extracted directly from yeast. To better purify the intermediaries, they were treated with exonuclease and digested with Not I. This was in order to remove any remaining linear yeast DNA. The DNA was then recombined into the complete genome. Once the genome is successfully transplanted, the new cell line can begin creating the desired proteins. The methodology outlined above is an example illustrating the potential applications Synthetic Genomics can contribute to bettering our environment. The company can successfully produce an artificial genome and transfer it to other living organisms. In effect, the company can efficiently design organisms that produce biofuels more efficiently, which is a necessary component in overcoming the multitude of current problems facing our environment.

Wed Dec 07 00:17:00 2011 Nayak Akanksha [email protected]

Molecular Staging Company:

Molecular Staging Technology: Rolling Circle Amplification Technology [RCAT(TM)] Molecular Staging formed an alliance with Motorola for the development of bioarrays for the detection of proteins[1]. This detection is carried out using the Rolling Circle Amplification Technology[RCAT(TM)]. This is used for the detection of disease molecules like DNA, RNA, proteins, antibodies and gene sequences and is especially good for detection of those molecules that are present at extremely low levels. This is beneficial for detection of diseases at their early stage. This method can be used to detect the presence of biomarkers, which can be either the target or a signal that is attached to the target[2]. In this method[linear RCAT(TM)] an oligonucleotide probe is used, which can attach specifically to the desired target. The other end of this probe is hybridized to a generic circular DNA template. This circular DNA template can be amplified by the technique of Rolling Circle amplification. Deoxynucleotides and a special DNA polymerase(ϕ29) are added to ensure that the template is amplified repeatedly to form multiple copies of the template DNA. The DNA that is produced in this manner can be labeled with multiple fluorescently labeled oligonucleotide probes [dinitrophenol (DNP) fluorescent tags]. Such a fluorescently labeled DNA molecule can then be condensed to a small point on the target using a multivalent cross-linking antibody against DNP. Multiple samples of potential targets can be assayed simultaneously by using different fluorescently colored labels on different oligonucleotide probes. This method can be used for target detection and validation in liquids, on solid phase(microarrays) and for direct amplification in cells and tissues. The advantage of this technique of target detection is that template and probe remain covalently attached to the target[2]. Microarrays have a variety of clinical applications. When used in conjunction with RCAT™, it can be used in the diagnosis of a variety of diseases. One scenario where this is used is in the detection of allergen specific IgE. The normal methods used for determining allergen specific IgE include skin prick testing and other invitro assays. These however have an equal chance of giving false positive results. Also, most of the invitro assays are semiquantitative and give results over a period of time. A modification of RCATTM called immunoRCA can be used for a more sensitive detection and measurement of proteins. In this method, the 5’end of the oligonucleotide primer is attached to the antibody. This is done in the presence of circular DNA, DNA polymerase and nucleotides. Concatamers of circular DNA are obtained which remain attached to the antibody. This amplified DNA is detected by fluorescently labeled oligonucleotide probes. Multiple allergen samples can be screened using this this technique on solid phase microarrays. The advantage of using this for allergen specific IgE over previously used techniques is that no false positive results were obtained[3]. Overall RCAT(TM) is an advantageous technique for sensitive detection of proteins(upto sub pg/ml can be detected). Targets can be located at their specific cellular location. This method can also be used to study the role of proteins and nucleic acids in disease and drug response. The method is quick, consistent and cost-effective. Further, since isothermal temperatures are used, cell morphology is conserved[4]. References: 1.Motorola and Molecular Staging Inc. Form Alliance to Develop Bioarrays; Companies to Jointly Research and Develop DNA, RNA and Protein Bioarrays. (19 December). PR Newswire,1. Retrieved December 3, 2011, from ProQuest Newsstand. (Document ID: 65959405). 2.http://www.molecularstaging.com/rcattm-technology-details-2.html (accessed 12/03/2011) 3.Mullenix, M. C., S. Wiltshire, et al. (2001). "Allergen-specific IgE Detection on Microarrays Using Rolling Circle Amplification: Correlation with in Vitro Assays for Serum IgE." Clin Chem 47(10): 1926-1929. 4.http://www.molecularstaging.com/rcattm-advantages.html (accessed 12/03/2011)

Wed Dec 07 00:34:18 2011 Kalin Alexander [email protected]

Oxitec

Oxitec RIDL Technology: A common problem with eradicating insects by pesticides is the possibility of toxicity to animals and humans. Currently, this issue has been solved by Sterile Insect Technique (SIT). SIT utilizes irradiation to sterilize insects and releasing the males over an extensive area. The males will mate with native females, but offspring will not survive, decreasing the females’ reproductive potential. After several releases over a substantial period of time, the pest population will be diminished or eliminated. SIT corrects the toxicity problem associated with pesticides, but it has several limitations. First, irradiation is damaging and weakening to the species, reducing the mating competitiveness of the released males. The released males cannot compete with native males in their weakened state; therefore, females produce viable offspring at a satisfactory rate to promote population survival. Second, it is difficult to separate sexes inexpensively and at a large-scale using SIT. It is important that only males are released because females are the source of damage, such as laying eggs on crops or extracting blood (mosquitoes). The technology developed by Oxitec, RIDL technology, resolves these troubling issues (1). RIDL technology involves inserting a dominant, lethal mutation into pests; however, RIDL pests survive normally if supplied with a specific supplement. The RIDL gene contains a tetO site, minimal promoter, and the tTA gene. Without a repressor, tTA is produced and binds to tetO binding sites. Binding to tetO increases tTA production, acting as a positive feedback loop. High levels of tTA are lethal to cells. A repressor, the supplement given to the insects, can bind to tTA, preventing tTA from binding to tetO. Only low levels of tTA are produced, which is harmless to cells (2). What makes RIDL technology so fascinating and useful is that the RIDL gene is heritable. RIDL insects for both sexes are constructed (supplied with the necessary supplement) and released (Bisex RIDL). After mating with native insects, any offspring die because they do not have the required supplement for survival. Consequently, sustained releases are necessary because the released pests will eventually die in absence of the supplement. This is also beneficial because RIDL insects that are accidentally released will not survive. Since the mutation is not weakening to the species, unlike using radiation, mating RIDL pests compete normally with natives (3). A major advantage of this technique is female-specific RIDL (fsRIDL), which utilizes alternative splicing. Splicing in females will produce the lethal gene, while differential splicing in males leaves them unaffected. Before release, the supplement is removed so that only fsRIDL males will be released. This eliminates the problem of mechanically separating sexes. After release, female progeny do not survive, suppressing the population. Continued releases are necessary, but eventually the pest population is unable to survive without females (3). Another advantage is that the insect strains are provided with a genetic fluorescent marker that can be seen under specific filters. Intriguingly, this marker is heritable, unfading, and non-toxic. Therefore, RIDL pests can be monitored and distinguished from native pests (4). References: 1) "Sterile Insect Technique." Oxitec. Oxford Insect Technologies. Web. 06 Dec. 2011. . 2) "Molecular Biology." Oxitec. Oxford Insect Technologies. Web. 06 Dec. 2011. . 3) "RIDL Technology." Oxitec. Oxford Insect Technologies. Web. 06 Dec. 2011. . 4) "Markers and Monitoring." Oxitec. Oxford Insect Technologies. Web. 06 Dec. 2011. .

Wed Dec 07 00:35:10 2011 Kim Caroline [email protected]

Biosearch

Technologies Messenger RNA (mRNA) detection is used for various purposes, some of them including determination of presence of specific transcripts in a cell, upregulated gene expressions, and differential gene expressions in different tissue samples (1). One common method used for mRNA detection is microarray. There are two major limitations to this method. First, quantification of the expression levels can only approximated from the signal strengths (1). Hence, accurate quantification of mRNA is difficult. Second, since mRNAs need to be extracted out from the cell before testing (1), localization of mRNAs on cells is not possible. Stellaris fluorescence in situ hybridization (FISH), a new technology developed by Biosearch Technologies, overcomes the shortcomings of microarray. Stellaris FISH is able to detect, localize, and quantify individual RNA molecules at the cellular level all at once (2). It utilizes labeled probes for RNA detection (2). What is unique about Stellaris FISH is that it uses a large number of probes to detect one RNA molecule (2). About 30 different probes (with the same fluorophore) targeting different parts of one particular RNA molecule are used (2). Using Stellaris FISH probes has two major advantages – one, due to a large number of probes used, at least one of them will bind to the targeted RNA (this means there is a high sensitivity); two, there is considerably low chance of having false negatives because it is unlikely for a “wrong” RNA to bind to more than one probe, hence giving out very small signal, which can easily be distinguished from the real signal (this means high specificity with low background) (2). The protocol for Stellaris FISH is very simple and can be summarized in four steps: (i) prepare and fix the sample on the slide; (ii) stain the slide with the Stellaris FISH probes; (iii) wash off any unbound probes; (iv) observe under the microscope and take the images (2). From these images, the number of transcripts in each cell can be counted directly with high accuracy and their locations within the cell can be determined (2). Furthermore, multiple transcripts can be detected at once by using mixtures of different probes with different colored fluorescence (so each mRNA is labeled with unique colors) (2). Stellaris FISH is a simple yet powerful assay that can be effectively used to detect, quantify, and locate specific transcripts of interest. References: 1. Dale, Jeremy W., & Schantz, Malcolm von. From Genes to Genomes (2nd ed.). West Sussex: Wiley, 2007. pgs. 313, 321-326. 2. Stellaris RNA FISH. 2011. Retrieved from http://www.biosearchtech.com/stellaris

Wed Dec 07 01:29:34 2011 CASTRO VERONICA [email protected]

Synthetic Genomics Inc.

Biotechnology has been around for over 10,000 years, its development goes from utilizing microbes to produce wine, bread, and cheese, to the creation of a bacterial cell controlled by a chemically synthesized genome (Synthetic Genomics). During the last decades, Scientists have been able to improve their ability to manipulate DNA using newly developed technologies such as recombinant DNA technology, allowing them to acquire a deeper understanding of genetics and proteomics involving a particular cellular process. One handicap of this method however, is that scientist have to start off with an organism’s genome and genetically modify it as much as the organism permits it. Synthetic Genomics Inc. is a privately held company founded in 2005, involved in the construction of gene and genome-length DNA using oligonucleotides. This company offers the research community a revolutionary technology in which any desired DNA sequence can be constructed allowing the synthesis of any gene or even entire genomes. Recently, Synthetic Genomics Inc. was able to transplant and express a chemically synthesize chromosome in a recipient cell. They used published genome sequence data to develop custom-design cassettes that encoded about 1080 bp of the DNA of interest; each cassette had an 80bp overlap to adjacent cassettes. These were then assembled together by transformation and homologous recombination in yeast (through multiple rounds). After verifying the sequence, this synthetic genome was transplanted into a cell (M. capricolum). The newly created cell presented with the expected phenotype and showed ability of continuous self-replication. Synthetic Genomics Inc. has made possible the creation of a synthetic cell by chemically synthesizing a genome and transplanting it into a recipient cell. Further more, they were able to prove that the transplanted synthetic genome was fully functional. This comes to show how synthetic DNA technology represents an important tool for the production of many useful applications (in research, and medical fields). The environmental field could also benefit by this technology; with today’s problem of global warming and with the continue growth in population the necessity for a renewable alternative energy source becomes essential. Currently, Synthetic Genomics Inc. is using this technology (synthesize completely new chromosomes that enable cells to performed a desired function) to engineer a strain of algae that could be refined to make biofuels. Furthermore, the ability of this technology to make small changes at the DNA sequence level can accelerate research for the production of vaccines and other drugs. In short, synthetic DNA technology provides a great potential for innovation and perhaps the answer to the development of new pharmaceuticals, biofuel sources, and other bio-based products. Reference Gibson, D. G. et al. Science 329, 52–56 (2010).

Wed Dec 07 10:11:34 2011 Strazzulla Lauren [email protected]

Cellectis

With the completion of the human genome project in 2001, there has been a considerable focus on genome engineering. This strategy relies on the ability to insert a gene into a specific location, and the specificity of this insertion is critical as inserting a gene at the wrong locus can have harmful if not lethal consequences. One of the most widely used techniques for genome surgery has been ZFNs (Zinc finger nucleases). However, there is considerable difficulty in custom engineering ZFNs with high enough specificity (1). In addition, engineering ZFNs has posed problems because this is both labor intensive and characterized by a high rate of failure (1). The development of TALENs by Cellectis (Romainville, France) has circumvented this problem resulting in a method for cost effectively altering the genome with an unprecedented level of specificity (2). Cellectis was founded on the vision of using nuclease engineering to make tools that will modify target genes (2). The company’s most successful contribution to genome customization has been a modified version of TALEs (Transcription Activator-like Effectors), which is a modulator protein with an N-terminal translocation domain, central repeats that mediate sequence specific DNA binding and a C-terminal segment that includes nuclear translocation signals and a transcriptional activation domain (2). TALEs are part of a highly conserved protein family originally found in Xanthomonas spp., which translocates into cells using a Type III secretion system (1). By altering TALEs to include a protein domain that can cleave DNA, Cellectis has created DNA scissors termed TALENs which are highly sequence specific (2). Genome editing using TALENs rests on the principle that nucleases can insert a DNA double stranded break which will activate DNA repair mechanisms resulting in knocking out genes or promoting gene targets (1). TALENs can cause gene modification in human cells by exploiting 2 main eukaryotic DNA repair pathways, NHEJ (non-homologous end-joining) and HR (homologous recombination) (1). Repair via the NHEJ mechanism often results in mutagenic deletions and insertions (1). Double stranded breaks can also result in homologous recombination between endogenous target gene locus and exogenously introduced homologous DNA fragments with the genetic information that is desired to be inserted (1). Since DNA damage repair mechanisms are highly conserved it is likely that TALENs could be effectively used among higher organisms such as plants, invertebrates, fish and mammals (3). Furthermore, Miller et al. (2011) reported successful use of TALENs for endogenous gene modification. Potential applications of this technology therefore include modifying the dose of a gene product, or decreasing the activity of nondruggable gene targets (4). Finally, Mussolino et al state that “it is conceivable that off-the shelf TALENs for each human gene will be available soon” (3). References: 1. Li, T., Huang, S., Jiang, W.Z., Wright, D., Spalding, M.H., Weeks, D.P., and Yang, B., (2010) TAL nucleases (TALNs) effectors and FokI DNA-cleavage domain. Nucleic Acids Reseach 39, 359-372. 2. http://www.cellectis.com/ 3. Mussolino, C., Morbitzer, R., Lutge, F., Dannemann, N., Lahaye, T., and Cathomen, T., (2011) A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Research doi: 10.1093/nar/gkr597. 4. Miller, J.C., Tan, S., Qiao, G., Barlow, K.A, Wang, J., Xia, D.F., et al., (2011) A TALE nuclease architecture for efficient genome editing. Nature Biotechnology 29, p. 143-148.

Wed Dec 07 10:35:17 2011 Kruchevsky Natalia [email protected]

Biosearch

Technologies Researchers are interested in finding out which cells express certain mRNAs and how many copies of mRNA are present in each cell. Currently, several methods exist to detect mRNA, such as northern blot, and real time- PCR. Northern blot has low sensitivity and only provides relative levels of RNA. RT-PCR requires a pure sample in order to work efficiently and the procedure is expensive. The Stellaris TM RNA FISH by Biosearch Technologies is a recently developed process that allows the simultaneous identification, and quantification, and location of mRNA. Among its many uses are the ability to analyze protein-RNA interaction, transcription site activity and mRNA translocation. This technique uses a four step protocol that can be completed in one day. The first step involved the fixation of the sample by permeablizing it with 70% ethanol. Then the probes are hybridized to the sample. Each probe is fluorescently labeled and is between 30-48 oligonucleotides. The probe hybridizes along the target mRNA. The use of large number of probes reduces the likelihood of false positive results. Positive signal is identified by the localization of many probes and detecting a signal above background. As a result, signal can be detected without the need for amplification. Weak binding generates a weak signal, bellow the florescence threshold. In the third step, the samples are washed with a buffer after four hour incubation time in order to remove any excess probes. The final step in the Stellaris assay is to image the sample using a florescence microscope. The assay can be used as a simplex, detection of one mRNA or multiplex, detection of several mRNA. The multiplex assay allows determining the relationship of expression levels between several gens simultaneously. In the multiplex assay several dyes are used at the same time. There are many benefits for the technology. One of the major advantages for this technology is that it generates high sensitive and high specificity of the sample due to the use of high level of probes. Another benefit is the ability of the method to visualize both cytoplasm mRNA and active site of transcription. Furthermore, the use of ethanol and not protease is used to fixate the cell, which allows the assay to be combined with florescence antibody assay. As a result, you can image both the mRNA and the protein it encodes at the same time. Finally, the technology provides a way to detect mRNA the method allows the detection of mRNA without the need to isolate or purify the sample from the cell. This method can be combined with other existing technologies such as immunohistochemistry, DNA FISH, qPCR and western blot. qPCR can be used after Stellaris RNA fish to confirm Alternative gene expression. It has potential to be used for drug screening in cells, by visualizing the up and down gene expression.

Wed Dec 07 11:32:48 2011 Neufeld Jessi [email protected]

Ion Torrent

Since the birth of DNA sequencing, most sequencing technologies have relied upon some type of optical detection method. Ion Torrent, however, has recently developed a novel strategy which bypasses the limitations of scanners, cameras, and light detection technologies. This innovative system relies instead on the detection of hydrogen ions, which are emitted during the addition of each successive nucleotide in the process of DNA synthesis. Interestingly enough, the company was founded by Jonathan Rothberg, who had previously made great strides in massively parallel sequencing when he founded 454 Life Sciences several years prior. Rothberg developed a microchip with a complementary metal-oxide semiconductor (CMOS), which has become increasingly affordable due to its continued reliance in the circuit manufacturing industry. The company hopes to soon reach its goal of a $1,000 genome with this new semiconductor sequencing technology. In this sequencing method, genomic DNA is initially fragmented, size-selected, and attached to 2.0 um acrylamide beads by way of adapters. These template DNA strands then undergo PCR amplification while attached to the beads in a step similar to other massively parallel sequencing techniques (such as Rothberg’s 454 sequencing). DNA polymerase and sequencing primers are then added, and this bead solution is spun in a centrifuge with a chip containing millions of 3.5 um-deep wells (which are only deep enough to allow the deposit of one bead per well). Nucleotides are then added to the wells sequentially (i.e. dCTP, then dATP, then dGTP, then dTTP), each for a duration of roughly 4 seconds (and then washed off before the next nucleotide is added). If a nucleotide is incorporated into the strand being synthesized, a proton will be released and will alter the pH of the well solution by a factor proportional to the number of nucleotides added in series. This change in pH is then detected by the underlying sensor and indicated in a digital readout, which provides sequencing information in parallel for each individual well. According to the Ion Torrent’s website (http://www.iontorrent.com/), this technology is capable of accurately producing up to 265-bp readouts with an error rate of only 2.99% at base 150 (as compared to a much larger error rate of 11.2% at base 150 using Illumina’s sequencing technology). Additionally, the company boasts a more complete coverage of the total genome (99.98%) than its competitor Illumina (94.17%). Ultimately, this technology shows not only a novel creativity, but also a level of exceptional accuracy and a promise of affordability that set it apart from other modern sequencing methods.

Wed Dec 07 11:37:06 2011 Agovino Eric [email protected]

Optogenetics

Optogenetics: For years, scientists have attempted to study how specific neurons affect behavior. The methods used for these studies were relatively crude and imprecise. For example, electrodes were inserted into neurons to stimulate them; however, the electrodes would typically stimulate too many cells, leading to imprecise results. Optogenetics, an important new technology that uses light to directly control living cells, overcomes this problem. This technology allows scientists to study the activity of specific cells in intact living animals (and observe the behavioral effects) with minimal invasiveness. As more fully set forth below, genes coding for light-sensitive proteins are introduced and expressed in cells, and light is then used to alter cellular behavior. There are many potential applications for optogenetics. In fact, optogenetics can likely be used to study almost any cell type or tissue. Accordingly, Nature called this technology Method of the Year in 2010, and Science dubbed it Breakthrough of the Decade. Optogenetics involves three basic steps: (1) identifying light sensitive proteins, (2) introducing the genes that encode these proteins to cells and expressing them, and (3) using light to manipulate cellular activity. With regards to step (1), the light-sensitive proteins can either be naturally occurring or chemically modified to become photosensitive. An example of a naturally occurring light-sensitive protein is channelrhodopsin, which is naturally found in green algae and serves as a light-gated ion channel. These light-sensitive transmembrane proteins are covalently bound to a chromophore (the moiety that absorbs light energy), which changes its conformation upon absorbing light, thereby activating the protein. Once activated, the ion channel opens and excites the neuron. In step (2), genes coding for these light-sensitive proteins are delivered to the target cells by transfection, viral transduction or the creation of transgenic animal lines. Expression can be restricted to specific cells using specific promoters. Alternatively, viral vectors can be used to target specific cells without specific promoters. In step (3), after the genes are introduced and expressed, light sources coupled to optical fibers can be used to shine light on exactly the right spot to activate (or inactivate) the cells of interest. Finally, behavioral testing can be used to assess the effect of modulating cellular activity in whole animals. One of the most common uses of optogenetics is to change the membrane voltage potential of cells. In neurons, membrane depolarization leads to action potentials, which are the basis of neuronal communication. Conversely, optogenetics can be used to cause membrane hyperpolarization, which leads to the inhibition of these signals. With these techniques, scientists can easily turn specific neurons on and off and then observe the effects on behavior.

Wed Dec 07 11:52:09 2011 Macri Vincent [email protected]

Nanosphere, Inc.

Verigene: Nano-particle assays for detection of nucleic acids and proteins. Nanosphere's Verigene technology allows testing of multiple nucleic acids (NAs) or proteins (or other epitope presenter) through application of a gold nanoparticle between 13-20 nanometers in diameter. This review will not describe Verigene’s proprietary apparati, but will instead describe detection methods. In human samples, it can be difficult to detect low-concentration NAs and proteins of interest. In addition, the time required for detection or diagnosis can be critical in cases such as infections of the blood. Nanosphere therefore attempts to address these issues by providing short-duration, signal-enhancing detection methods for NAs and proteins in human samples. Verigene (directed to prostate-specific-antigen assay) was shown to be 300 times more sensitive than other immunoassays. In addition, Verigene was able to detect 13 bacterial pathogens and their antibiotic resistance in 2.5 hours, as opposed to existing culture methods which may take one to 2 days. Nucleic Acid Detection: Nucleic acid (NA) detection is achieved using two sets of single-stranded oligonucleotides, each of which is complementary to a separate region on the target NA. One ss oligonucleotide (“capture oligo”) is fixed to a solid surface of a cartridge (as in a microarray), while the other (“signal oligo”) is fixed to a gold nanoparticle. The process of NA detection is completed in approximately 90 minutes, and proceeds as follows in Nanopshere’s proprietary cartridge and processor: Target NAs are sonicated to length 300-500 nucleotides and subsequently passed over the capture oligo. Hybridization occurs between complementary oligos and target NAs, and unbound NAs are washed away from the reaction mixture. The gold nanoparticle (with signal oligo) is then introduced; the signal oligo hybridizes with target NA free end. Unbound nanoparticles are then washed from the reaction mixture, leaving the following complexes: fixed surface--capture oligo--target--signal oligo--nanoparticle. Finally, signal amplification (500,000x greater than fluorophore) is achieved by binding silver to the gold nanoparticles. This eliminates the normal requirement for PCR amplification before detection, which increases assay duration and can be error prone. Under proper hybridization conditions, oligo-target binding specificity can be adjusted to detect a single mismatched base pair, allowing the method to be used to detect SNPs. Protein Detection: Protein detection is achieved through application of a magnetic nano-particle, to which is bound a primary antibody to the protein of interest, and a gold nanoparticle, to which is bound a secondary antibody to the same protein. Also bound to the gold particle are multiple, identical double-stranded nucleic acid (NA) "reporters" with sequence that is specific for the protein of interest. The process of protein detection proceeds as follows: The primary antibody (attached to magnetic nanoparticle) and the secondary antibody (attached to gold nanoparticle) bind the protein of interest. A complex is therefore created: magnetic particle--protein--gold particle. The complex is sequestered by applying a magnetic field across the solution, after which any unbound proteins and nano-particles are washed away, and the complex is re-suspended. The new solution contains a buffer which (along with increased temperatures) causes the double-stranded NA reporter (attached to gold nano-particle) to denature/dissociate. Multiple reporters dissociating from each gold nanoparticle provide signal enhancement of the bound protein. NA reporters can be collected and quantitively detected using the Nucleic Acid Detection process described above. If detection of multiple unique proteins is desired, multiple nano-particle designs are possible, each containing unique secondary antibodies and NA reporters. Verigene claims that most of its clinical diagnostic assays can be completed in 2.5 hours, with “hands-on time” of less than 5 minutes. Verigene therefore provides an automated system for short-duration, high-signal detection with reduced opportunity for user or experimental error. References: 1. www.Nanosphere.us 2. Bao YP, Huber M, Wei TF, Marla SS, Storhoff JJ, Müller UR. (2005) SNP identification in unamplified human genomic DNA with gold nanoparticle probes. Nucleic Acids Research 33(2):e15 3. http://www.ncbi.nlm.nih.gov/pubmed/19841273 4. http://www.jurology.com/article/S0022-5347%2810%2901090-6/fulltext 5. U.S. Patent # 7,259,252 http://patft.uspto.gov/netahtml/PTO/srchnum.htm

Wed Dec 07 13:17:13 2011 Yu Shui [email protected]

Affymetrix

Even 99% of the DNA sequences we share are identical, a small difference of the DNA sequence is still sufficient to give rise to serious effects on human health. For example, a miss-match mutation can disrupt the normal function of gene and cause malfunction and disease. Since there are 3.1 billion base pairs in human genome, finding the mutation or malfunction in one or more genes becomes a big challenge for researchers(1). The invention of DNA microarrays allows researchers to be able to scan the whole genome and identify the SNPs among patients in order to find their genetic similarities. By genotyping SNPs, the disease causing genes could be pinpointed. The GeneChip DNA microarray technique manufactured by Affymetrix used for genotyping SNPs, has been employed in approximately 24200 scientific publications so far(2). The manufacturing process of the GeneChip DNA microarray involves photolithography, where the light is used to selectively activate the chip surface, and chemical reactions with the DNA blocks would take place as a result. This selective activation process undergoes repeated cycles and is in predetermined patterns, which allows numbers of different DNA probes to be built up in parallel and gradually(3). One main advantage of the GeneChip microarray is that instead of just doing one SNP each time, more than 100,000 SNPs can be scanned at once(1). This is because the size of the feature containing each type of DNA probe is extremely small, and as the feature size is reduced, the amount of information contained on single array increases exponentially(3). In addition, there are more features than SNPs, which makes sure that the location of each SNP will be identified correctly, since numbers of different probes are applied to measure the genotype of each SNP on the array(1). A test sample can be introduced into the DNA probe array where hybridization occurs. Each probe on the chip is in the form of single strand DNA and hybridization would take place when two complimentary strands of DNA come together and form a complex. When a single strand DNA test sample is washed over the surface, the single strand sample will bind to its complimentary DNA probe. Each single strand DNA in the test sample is tagged with biotin, which will bind to the fluorescent molecules in the fluorescent staining step later. After the hybridization, unbound single strand DNA sequences will be washed away and the hybridized features will be coated with biotin. Then the array is washed by fluorescent stain and under the laser light, fluorescent signals could be observed at places where hybridizations occur(1). Since the location of each probe is already known, by using the Affymetrix GeneChip Operating software, researchers can identify the DNA sequence in the sample and determine the presence of the target gene and whether or not there are mutations on that gene or group of genes. The mission of Affymetrix is to “revolutionize how the world benefits from genetic information” (2). Affymetrix microarray technology helps researchers discover genes behind many diseases and enables the development of personal treatment. Reference: 1. Affymetrix. GeneChip® Microarray Student Manual, student activity 2: Overview of GeneChip® Microarray Structure and Function. http://www.affymetrix.com/about_affymetrix/outreach/educator/microarray_curricula.affx#1_2, Accessed at December 7th, 2011. 2. Affymetrix. About Affymetrix. http://www.affymetrix.com/estore/about_affymetrix/index.affx?category=34022&categoryIdClicked=34022&rootCategoryId=34003&navMode=34022&parent=34022&aId=aboutNav, Accessed at December 7th, 2011. 3. Affymetrix. GeneChip® Microarray Student Manual, student activity 3: Photolithography: How GeneChip® Microarrays are Manufactured. http://www.affymetrix.com/about_affymetrix/outreach/educator/microarray_curricula.affx#1_3, Accessed at December 7th, 2011.

Wed Dec 07 13:32:58 2011 Olguin Arturo [email protected]

Ion Torrent

We chose this company because as we saw in class different types of sequencing technologies, and how they are getting close to the one thousand dollar genome project, we discovered this company that uses a very interesting technology to do genome sequencing. Ion Torrent uses two types of machines. One is a chip which is no different from the chip included in smart phones like iphones or blackberries. This chip has on top multiple wells (depending on the model up to 12 million) where the sequencing reaction will take place. Below the wells it has an ion sensitive layer which will detect any ions released into the media. And on the bottom of the chip there is a proprietary ion sensor which will translate the chemical data (pH changes) to digital data. The second machine they use is a Personal Genome Machine (PGM) Sequencer, which will provide the chip with all the reagents necessary for the sequencing reaction to happen. How does the whole thing work? First, as some other second generation sequencing techniques, it involves a library preparation of the genome sample. Once the genome has been broken into small templates (~100 - 400nt, depending on the chip used), using dilutions a single template is attached to a bead and amplified all over the bead. Each bead containing several copies of the template is placed in a single well on the chip. The PGM will now introduce all the reagents needed for the sequencing reaction to take place. One of the key differences and advantage of this technique is that it uses natural, unmodified nucleotides. One nucleotide is added at a time (T – A – C – G ), with a washing step between each nucleotide addition. Naturally, during the normal DNA polymerization reaction, each time a nucleotide is added, a hydrogen ion (H+) is released into the media, lowering the pH. These changes on the pH will be detected by the ion-sensitive layer and directed to the ion sensor which will interpret the voltage changes and translate the signal into a digital graphic. Each time a nucleotide is added a measured voltage change will be shown in the computer; if two or more nucleotides are added (e.g. two adenines) the voltage change will be doubled or multiplied since the voltage measurement is almost linear. Of course with poly nucleotide regions, the accuracy of detection lowers, since it is not completely linear. Each time the PGM adds a nucleotide, there is a 7 second window to detect all the voltage changes signals, after that there is a 30 second washing period and the second nucleotide is added. This theoretically allow us (depending on the chip) to have complete experiments sequenced in approximately 2 hours. I think this is a very interesting approach to the sequencing reaction that is independent of modified nucleotides, expensive laser or light detection machines or picture taking machines. By using simple pH detection, the cost is reduced significantly and the sequencing time is even shorter since the DNA pol is working as fast as it would normally work.

Wed Dec 07 13:43:35 2011 Lu Yang [email protected]

Nanosphere Inc

Nanosphere Incorporation is a company that produces the innovative technology for clinical diagnosis. The reason we chose to discuss the company is because of their core technique¡ªgold nanoparticle. The gold nanoparticles work as a secondary hybridizing factor of either a probe or an antibody, which is unlike the probes and antibodies that we have discussed in class. The probes and antibodies that we discussed before only primarily hybridize to their target sequence to show the presence of the target, while the gold nanoparticles with short oligonucleotide sequences or protein sequences bind to the probes or antibodies that have already bind to the target. Only when both the hybridization between the nanoparticle sequence and the probes or antibodies, and the hybridization between the probes or antibodies and the target sequence bind perfectly, a signal can be detected. The gold nanoparticles are roughly 13~20 nm in diameter, and they either function with a defined number of oligonucleotides with short sequence complimentary to target sequences, or a defined number of antibodies. The gold nanoparticles have several properties which make them a good tool for detecting a target. Firstly, the gold nanoparticles have high sensitivity. The signal emitted from the gold nanoparticles is 5*105 times than a regular fluoropore. Secondly, the gold nanoparticles have high specificity. They can detect a single base pair differentiation. Thirdly, the nanoparticles have less background noise interaction with less non-specific binding to the target. Thus, the accuracy of the result is increased. Lastly, the gold nanoparticles are very stable and non-toxic, so they provide a safe detecting assay. The company has widely applied the gold nanoparticles clinically. In identifying the infectious disease, the gold nanoparticles make the identification of infectious microbial more rapid, so the patients can receive proper treatment faster. In personalized drugs, the easy way of detecting SNPs by the gold nanoparticles offer a better therapy decision based on each patient individual. In cardiology, the gold nanoparticles facilitate in diagnosing the non-specific symptom which is a most challenging problem when diagnosing cardiology diseases. With the gold nanoparticles, the diagnostic and treatment decisions of a cardiology patient become more efficient. In human genetics, the improved way to detect a certain gene with the gold nanoparticles can assist physicians in making better decision in patient¡¯s risk of developing a disease or the patient¡¯s potential response to treatment. In summary, the Nanosphere Inc. has developed the technique utilizing the gold nanoparticles in wide range of clinical application. All these application reflects the improvement in the assay of direct detection of nucleic acid and high-sensitivity protein diagnostics. However, the gold nanoparticles are not perfect. A severe side effect is that nanoparticles can induce DNA damage across a cellular barrier (Bhabra et al. 2009). With the pitfall in mind, the gold nanoparticles technology can be further improved. Reference Bhabra G., Sood A., Fisher B., Cartwright L., Saunders M., Evans W. H., Surprenant A., Lopez-Castejon G., Mann S., Davis S. A., Hails L. A., Ingham E., Verkade P., Lane J., Heesom K., Newson R., and Case C. P. (2009) Nanoparticles can cause DNA damage across a cellular barrier. Nature Nanotechnology (1038): 313-321

Wed Dec 07 13:43:48 2011 Lu Yang [email protected]

Nanosphere Inc

Wed Dec 07 13:43:54 2011 Lu Yang [email protected]

Nanosphere Inc

Wed Dec 07 13:46:21 2011 Nik Sara [email protected]

Ion Torrent

Ion Torrent, a unit of Life Technologies, is a biotechnolgy company that utilizes integrated semiconductor technology to carry out non-optical DNA sequencing. Ion Torrent released their semiconductor sequencing technology in February 2010 and has since sought to market their machine as a more rapid, compact and affordable option for labs of all sizes, compared to other next-generation DNA sequencers. Prior to the development of semiconductor based sequencing, DNA sequencing has been “limited by its requirement for imaging technology, electromagnetic intermediates, and specialized nucleotides or other reagents.” (1) Ion Torrent however is the first company to make a ‘post-light’ sequencing technology commercially available. Unlike light-based DNA sequencing technologies, Ion Torrent’s sequencing technology does not rely on optics, lasers, cameras, or fluorescence to provide sequence readout. Instead, this instrument is comprised of a disposable ion semiconductor chip, software for signal processing and a fluidics system to control the flow of reagents over the sensor. The massively parallel, proprietary semiconductors sensors output sequence data in real-time by detecting the ions released during template-directed DNA replication. The sequencing mechanism utilized is straightforward. As opposed to previous technologies based on sensors designed to detect photons, here the DNA sequence is determined by measuring the hydrogen ions released (1 per base added per DNA strand) during second strand synthesis when DNA polymerase sequentially adds complementary bases. When the hydrogen ion is released, it produces a shift in the pH of the surrounding solution proportional to the number of nucleotides released (i.e. if there is homopolymer stretch). This shift in pH is then detected by a sensor, which is converted into a voltage, and digitized into a base call via signal-processing software. Since this real-time detection of hydrogen ions is a natural process, the sequencing chemistry uses unmodified nucleotides and native enzymes. The ion semiconductor chip bridges a direct connection between the biological information in the DNA and the digital information in the sequence readout. In terms of run quality, its accuracy “is similar at 50 bases and higher at 100 bases than light-based method using modified nucleotides (1.1% versus 5% error)” (1). Ion Torrent semiconductor based sequencers also make DNA sequencing a scalable and low-cost technique. Their proprietary design makes use of the complementary metal-oxide semiconductor (CMOS) process for building their integrated circuits. A benefit of this is that CMOS are already a widely used, low-cost industry standard technology found in common technology such as personal computers, digital cameras and cells phones. As a result, semiconductor sequencing is a highly scalable process since the technology is readily available; users can match the size of the chip to their specific application Applications for the Ion Torrent semiconductor include small genome sequencing, whole genome sequencing, RNA-seq, ChIP- seq and targeted re-sequencing, among others. Essentially, Ion Torrent aims to make it affordable and convenient for individual labs to have their own sequencing instruments, and to work further towards achieving the goal of the $1,000 genome. References: 1) Rotherberg JM et al. An integrated semiconductor device enabling non-optical genome sequencing. Nature. 2011 Jul 20;475(7356):348-52. doi: 10.1038/nature10242.

Wed Dec 07 15:22:06 2011 Leon Katerina kl2617

Dendreon

Dendreon Corporation Prostate cancer is an important cause of morbidity and mortality in men in the United States, being the most diagnosed and the second cause of death due to this disease after lung cancer (4). When the cancer is still localized it can be treated with radiation therapy or surgery but in 20-30% of the patients it becomes metastatic and is mostly treated with hormone therapy which uses drugs that antagonize the action of androgen molecules that act through the androgen receptor, a receptor expressed in high levels when cancer becomes metastatic (5). Hormone therapy can be very effective but in most men the disease progresses to a most aggressive form called metastatic castration-resistant prostate cancer in which the only treatment available is chemotherapy yielding a median survival of 12 to 21 one months approximately (2). Dendreon Corporation has recently developed a new type of treatment called autologous cellular immunotherapy to treat metastatic castration-resistant prostate cancer. This type of treatment takes the patient’s own dendritic cells and activates the immune system to fight the disease. This immunotherapy approved by the FDA in 2010 with the name of Provenge increases the patient’s life expectancy in 4 months with minor side effects compared to those of chemotherapy (1). The first step in Provenge treatment involves taking patient’s mononuclear cells in peripheral blood through a leukaperhesis procedure. These autologous mononuclear cells include antigen presenting cells (APCs), B cells, T cells and natural killer cells among others. Provenge manufacturing uses APCs, which are activated by fusion with a recombinant human protein. This recombinant human protein consists of an antigen expressed in prostate cancer tissue called prostatic acid phosphatase (PAP), and an immune cell activator called granulocyte-macrophage colony-stimulating factor (GM-CSF). Once APCs are fused with PAP-GM-CSF through ex vivo culture, APCs take the recombinant fusion protein and present it as small peptides on their surface. Then Provenge is infused into the patient where it will trigger a tumor directed immune response by the natural function of APCs of activating T cells, which will now be targeted against PAP (3). Every Provenge dose roughly contains 50 million CD54+ cells, which are surface molecules used to determine APC activation, activated with the recombinant protein PAP-GM-CSF suspended in 250 mL of Lactated Ringer’s injection. Each patient will require 3 doses of Provenge during a one-month period, in which patient’s peripheral blood will be collected 3 days before each infusion (3). This novel approach represents a major breakthrough by being the first form of targeted or “personalized” medicine against cancer approved by the FDA, and opens the door to the discovery of more candidate molecules that can activate the immune system to fight other diseases. Dendreon Corporation represents a very interesting and promising biotechnology company because through active cellular immunotherapy (which brought Provenge to the market), other candidate molecules against different types of cancer like kidney, colon and cervical cancer are currently being developed (1). Rereferences 1. Dendreon, 2011. Taken from http://www.dendreon.com/ on December 7, 2011 2. Kantoff, P., Higano, C., Shore, N., Berger, R., Small, E, et al., (2010). Sipuleucel-T Immunotherapy for Castration-Resistant Prostate Cancer. N Engl J Med 363 (5): 411-422 3. Provenge, 2011. Taken from http://www.drugs.com/pro/provenge.html#s29 on December 7, 2011. 4. Rosenthal, S., Sandler, H. (2010). Treatment Strategies for High-Risk Locally Advanced Prostate Cancer. Nat Rev Urol. 7(1):31-38 5. Tran, C., Ouk, S., Clegg, N., Chen, Y., Watson, P, et al., (2009). Development of a Second-Generation Antiandrogen for Treatment of Advanced Prostate Cancer. Science, 324(5928): 787–790.

Wed Dec 07 15:54:29 2011 Song Ziwei [email protected]

TwistDx

PCR is a conventional DNA amplification method, routinely performed in a precisely temperature-controlled apparatus with repeatedly cycle through hot and cold phases, which restricts the marvelous applications of PCR within experimental laboratories and sophisticated professionals. Scientists from TwistDx have developed a revolutionary technology of DNA amplification that overcomes the technical difficulties of conventional PCR technique, called Recombinase Polymerase Ampliﬁcation (RPA). With no requirement of thermal denaturation, RPA can be carried out at a constant low temperature (37℃) with a probe-based detection technology, using only a tiny amount of DNA, without elaborate equipments. RPA employs five main ingredients: A. a target DNA template to be ampliﬁed; B. a primer–recombinase complex, initiating the elongation process after combining to the template; C. nucleotides to form the new DNA strands; D. a polymerase to synthesize new DNA strands; E. single-stranded DNA-binding proteins (SSBs) prevent template DNA from zipping back together. The amplification of RPA does not require global melting of the template to direct primers to complementary sites target sequence. Instead, RPA employs recombinase-primer complexes to scan along double-stranded DNA and facilitate strand exchange at cognate sites [1], attaching to the double-stranded DNA, eliminating the need to heat the mixture. After recombination, recombinases disassemble from the 3’-end of the oligonucleotide, allowing the DNA polymerase to begin synthesizing a new strand of DNA complementary to the template, while the SSBs attach to and stabilize the displaced strand. The DNA amplification process automatically repeats, resulting in an exponential increase at a constant low temperature in a short period (Fig. 1). The key point of RPA is a precise dynamic reaction environment of process-regulating chemicals, which balances the formation and disassembly of recombinase-primer complex. With the presence of ATP, recombinase binds to oligonucleotides, forming nucleoprotein complex that hydrolyze ATP into ADP, which then resulting recombinase disassembly and integration of polymerase. Scientists from TwistDx have established a favorable and precise reaction conditions with recombinase (T4 uvsX), SSBs (T4 gp32) and crowding agent (uvxY Carbowax20M) (Fig. 2). Except for broad application in DNA amplification and detection, the reaction environment of RPA improved understanding of recombination machinery and DNA hybridization. RPA has significant advantages of portable and accessible nucleic acid–based testing compared to PCR. It has also been utilized to test the presence of methicillin-resistant Staphylococcus aureus (MRSA), known as “Superbug”, leading to rapid, accurate and sensitive outcomes that detected fewer than ten copies of MRSA DNA with distinguishing presence of three different genotypes [2]. Compare to other diagnostics methods, RPA is rapid, portable and accessible with exquisite sensitivity and specificity. With the massive capability of amplifying DNA in normal environment, the technology of RPA is widely applicable in medical diagnostics and health testing. Reference: [1] Olaf Piepenburg, et al. , DNA Detection Using Recombination Proteins. Plos Biology, July 2006, 4 (7) e204 [2] Siobhan Wagner, Infection detection: Testing device uses an RPA technique to determine the presence of the MRSA virus in patients. The EnGIneeR 9–22 FEBRUARY 2009

Wed Dec 07 16:12:12 2011 Wang Chen-Lin [email protected]

Cellectis

Cellectis is a biotech company located in Paris, providing genome customization techniques causing targeted chromosome integration, modification and disruption. These techniques involve in a specific DNA scissors enzyme called meganucleases and the use of natural repair mechanisms. Meganucleases are designed based on a transcription activator–like (TAL) effectors. The TAL effectors protein can recognize specific DNA sequences via a 33-35 amino acid repeat region. What makes it useful is that the DNA recognition by the TAL effectors protein is based on a predictable DNA sequence. Therefore, the TAL effectors protein can be modified to recognize virtually any DNA sequence. TALEN method fuses the TAL effectors protein with the catalytic domain of an endonuclease, making a sequence-specific class of nucleases. Based on TAL effectors, meganucleases are engineered to recognize and cut virtually any DNA code, and the long recognition site makes them unique and highly specific nucleases. After the introduction of DNA double strand breaks (DSB) by meganucleases, cells will repair DSB through Homologous Recombination (HR) or Non Homologous End Joining (NHEJ). In the DNA repair system, homologous recombination makes template DNA carrying an additional transgene incorporated into chromosome, resulting in transgene targeted integration at the meganuclease recognition site (Fig 1a). Also, the template DNA containing a mutant genomic DNA can also be applied to make targeted gene modification (Fig 1b). Furthermore, non-homologous end joining (NHEJ) makes the re-ligation of two double stranded DNA. After treating with addition or deletion nucleotide stretches raging from one nucleotide to several kb, the NHEJ process can be used for targeted disruption (Fig 1c). The following are key advantages of meganuclease targeted integration and disruption. 1) It provides predictable transgene expression. In traditional transfection method, researchers use random integration thus cells will always express different levels of protein. With the kit, they will have a single copy of gene. Also, the integration is performed in a single selected genome site. 2) Little clone screening. High clone homogeneity (at least 95% targeted integration) is obtained because of the efficient integration method. 3) Highly stable protein expression. The gene is integrated a hot-spot locus selected for very high expression. This method guarantees the production of target proteins at a very high level and good stability even in the absence of selection agents. 4) Rrobust data comparison and homogeneity. Because the generated clones are isogenic, drug profiling on these cell lines is much more robust. In addition, since the clones are homogeneous, researchers do not need to screen a lot of them. As they called this product “cellular Genome Positioning System” or cGPSR, it makes researchers know where the gene insertion occurs. This technology is a great progress in science and technology.

Wed Dec 07 17:28:23 2011 Pradhan Radhika [email protected]

TwistDx

Recombinase Polymerase Amplification By TwistDx Kary Mullis’ simple idea of a process that involves iterative amplification of DNA so that substantial amounts may be obtained for easier and thorough analysis was a revolution in the field of molecular biology. This process, called Polymerase Chain Reaction or PCR, involves cyclic denaturation, amplification and extension of the two DNA strands, each held at varying temperatures for a certain number of cycles. [1][2] It paved the way for breakthrough research in almost all areas of scientific research, ranging from the bench to the bed- e.g. genotyping, pharmacogenomics, etc. to disease analysis. However, this process is confined to sophisticated research and diagnostic labs that have the equipment to carry out this reaction (since it is heavily heat dependent), and the funding to afford all the costs associated with the reagents and enzymes as well as their maintenance. TwistDx has come up with a technology which overcomes these obstacles. Called Recombinase Polymerase Amplification (RPA), it is an isothermal PCR that exploits the ability of recombinases to detect the presence of specific sequences on the parent DNA strand and give exponential amplification by strand-displacement DNA synthesis. RPA uses five ingredients: 1. DNA sample to be amplified, 2. A primer–recombinase complex that detects the presence of a specific sequence on the template DNA 3. Nucleotides for incorporation into the growing strand 4. A polymerase for incorporating the nucleotides, and 5. Single-stranded DNA-binding proteins (SSBs), which maintain the DNA replication bubble. The primer–recombinase complex attaches to the double-stranded DNA at a constant temperature of 37 oC. DNA polymerase then begins synthesizing a new strand of DNA complementary to the template in a D loop, while the SSBs stabilize the displaced strand. This process takes place in the presence of proprietary regulatory chemicals and reaction-controlling reagents. After the polymerase is done replicating the template DNA, this process repeats. [3] This technology has several advantages. The reactions are sensitive, specific, fast and isothermal at a low temperature (37 oC). It has been proven to detect less than 10 copies of DNA in for the detection of Methicillin Resistant S. aureus. It does not require any sample preparation. [3] It takes 5-10 minutes to go from 1 copy to detectable levels of DNA. Also, the reaction system is robust, and has been developed to be a dried formulation that does not require refrigeration. [4] This technology brings PCR into fieldwork. A sandwich assay has also been developed to encapsulate the entire system in a dipstick, and this can be used for pathogen detection. [5] Also, it brings PCR to the masses due to significantly lowered costs and relative ease of use. References: [1] Shampo MA, Kyle RA. “Kary B. Mullis--Nobel Laureate for procedure to replicate DNA.” Mayo Clin Proc. 2002 Jul;77(7):606. [2] Kary Mullis Nobel Lecture, December 8, 1993 [3] Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA Detection Using Recombination Proteins. PLoS Biol 4(7): e204. doi:10.1371/journal.pbio.0040204 [4] TwistDx Technology. http://www.twistdx.co.uk/our-technology/page_10.html Retrieved: 11/27/2011 [5] Mary Hoff “DNA Amplification and Detection Made Simple (Relatively)” PLoS Biol. 2006 July; 4(7): e222. Published online 2006 June 13. doi: 10.1371/journal.pbio.0040222

Wed Dec 07 18:15:22 2011 Dendy Meaghan [email protected]

Oxitec

Wed Dec 07 18:30:43 2011 Xu Luyao [email protected]

Affymetrix

Affymetrix is a biotechnology company that is currently the leading provider of microarray technology to research labs. Their microarray technology involves the use of a basic idea that nucleic acids can complement each other and form base-pair when hybridized. In brief, an organism is consist of many different types of tissues, depend on the external environment and stage of development, cells in a tissue may express or suppress specific gene transcription and thus alter the relative level of gene products such as mRNAs and proteins. Microarray technology offered by Affymertix is aimed to compare two different populations of cells or types of tissues on their gene expression level. The mRNA abundance of a cells or tissue is measured and gene expression profiles are generated. For example, we can analyze the difference of gene expression profiles between cells taken from cancerous tissue and normal tissue to determine genetic changes of two populations. Once gene expression alterations are detected, then we may be able to manipulate these genes to control onset or progression of cancer. Gene chip is essential for Affymertix microarray technology, where oligonucleotides probes that represent individual genes were synthesized on a glass slide. Multiple oligonucleotide probes are synthesized to cover the entire genome. Single stranded probes represent individual gene are made in a small area on the chip to hybridize complementary sequence. And collective anchored oligonucleotide probes upon binding of the labeled cRNA of cells can give off fluorescence signal. To prepare the microarray, mRNA is extracted from cells and reverse transcripted into cDNA. Then cDNA is in vitro transcripted to cRNA where is then labeled with biotin. Subsequently, cRNA is fragmentated to short strands and the single strand fragmented biotin-labeled cRNAs from different samples are mix together. Then, the mix is allowed to hybrdize to the chip overnight. After removal of non-hybrdized molecules, biotins on cRNA are fluorescently labeled. Probe and single strand cRNA will generate signal that can be detected by scanner. Computer is used to create and keep track of different fluorescence spots on the gene chip. Overall data collectively represents the relative gene expression of cells. Instead of looking at one individual gene changes, Affymertix’s microarray provided an effective method to look at the genetic changes in the whole genome of an organism on one chip. We can utilize these information to understand how genes work together to yield a specific phenotype. Furthermore, Affymertix does not only offer microarray to detect gene expression, the company also apply the same methodology to detect changes in genomic region of genes as well as SNPs in DNA sequence. In conclusion, Affymertix offers a very powerful and efficient technology to screen many gene changes. As the technology become mature, Affymertix microarray will undoubtly become widely used in many laboratories.

Wed Dec 07 20:02:48 2011 Lin Edwin [email protected]

Dendreon

Dendreon Corporation is a Seattle-based pharmaceutical biotechnology company. Its flagship drug is Provenge, an immunotherapy treatment against late-stage metastasized prostate cancer. Before Provenge, there was no effective treatment for prostate cancer after metastasis. The traditional method of treatment for these late-stage patients consisted of either hormone therapy or chemotherapy. It is known that testosterone is required for prostate cells, as well as prostate cancer cells, to grow. Therefore, suppressing testosterone levels in the body would decrease the growth of prostate cancer. Testosterone can be suppressed by medication such as leuprolide (Lupron) or histrelin (Vantas). Other methods of testosterone suppression include surgery to remove the testicles. However, suppressing testosterone has many side effects, including loss of bone and muscle mass, and has been linked to cardiovascular problems. Another side effect of surgery is nerve damage. Resistance to hormone therapy is not uncommon, and some prostate cancers are also castration-resistant. Chemotherapy is also used as a last resort to late stage prostate cancer. Because cancer cells replicate very quickly, drugs that inhibit cell division or damages cell nuclei can be used to target the prostate cancer. Although it can kill cancer, it also kills other cells that divide quickly, such as hair, skin, and bone marrow cells. Killing these cells lead to very severe side effects for the patient and thus, chemotherapy is used as a last resort for patients who have developed resistance to hormone therapy. Provenge (approved in 2009) has been shown clinically to have minimal side effects, while increasing life expectancy by four months compared to traditional treatments. Provenge is based on using the patient’s own immune system in order to target cancer cells. Normally, the human immune system attacks infections with T cells, which are activated by antigen presenting cells (APC). APCs endocytose antigenic proteins, digest them into fragments, and present them on their cell surface. T-cells, which can recognize these antigen fragments will then be activated and attack cells bearing the antigen. Provenge exploits this mechanism to fight cancer. A patient’s APCs are extracted at a clinic and sent to the company to be activated. The patient’s APCs are exposed to prostatic acid phosphatase (PAP), which is an antigen expressed in 95% of prostate cancers. The APCs will take up the PAP and fragment the protein, which will then be presented on the surface of the APCs. The APC is also given granulocyte macrophage colony-stimulating factor (GM-CSF), which is an immune cell activator. Exposure to these compounds will activate the APC cells, which will then be injected back into the patient. T cells can then be activated against the PAP antigens present on the APCs, which will allow them to recognize and attack prostate cancer cells. Thus, Provenge is a safer, more effective treatment for late stage prostate cancer compared to hormone therapy or chemotherapy.

Wed Dec 07 20:03:02 2011 Lin Edwin [email protected]

Dendreon

Wed Dec 07 23:40:37 2011 McCray Jacqueline [email protected]

Molecular Staging, Inc.

Together, Molecular Staging, Inc. (acquired by Qiagen NV) and Amersham Pharmacia Bio-tech have created Rolling Circle Amplification Technology (RCAT). Although, very similar to Rolling Circle DNA synthesis its main goal is not to sequence DNA. Instead it is used to magnify segments of DNA, in order, to help identify unknown pieces of DNA, RNA, or protein from different viruses, cells, antibodies or other sources [1]. This amplification is sometimes needed when biomarkers of interest are present at extremely low levels. With this in mind, RCAT can be used in both the clinical and research settings. RCAT has been used to amplify vector DNA [2], to amplify viral DNA genomes [3], and to even profile up to 150 proteins in a number of different substrates, such as serum, plasma and supernatants [4]. RCAT negates the need for bacterial colonies and the step-wise approach, sometimes needed, to diagnose disease or other conditions. RCAT can be used with several widely used technologies, such as Luminex bead assay systems. In addition, it can be used on microarrays, where amplification can take place right on the slides, eliminating the need for PCR [1]. There are three types of RCAT: linear-RCAT, exponential-RCAT and multiply primed-RCAT [1]. The linear RCAT works by using a DNA probe that is attached to both a universal DNA circle and the biomarker of interest. Phi 29 polymerase is used to replicate the circle continuously, because of its high fidelity [5] . When complete, there are hundreds or even thousands of copies of the DNA circle, connected in a single strand. Fluorescent dyes are then used to attach to the many available binding sites [1]. RCAT can also be used inside the cell, which helps to increase fluorescence in many existing technologies, such as in-situ hybridization [1]. In other situations, since it can take place inside the cell, replication can proceed without altering any bound protein’s structure, which can occur with increased temperature created during other replication methods, that utilize thermal cycling. Exponential-RCAT takes it one step further. The result is that, by using a second DNA probe, both the circle and the growing RCAT product are amplified. The last type of RCAT, multiply primed-RCAT takes advantage of several primers attached to the circular universal DNA template. Thus, in all three types of RCAT you are left with a multitude of fluorescent tags to help identify your product of interest or simply amplified DNA that can later be used for other purposes, such as sequencing, if needed. Currently, GE Healthcare holds the commercial license for TempliPhi(TM) that is based on RCAT [6]. References: 1. Molecular Staging, Inc. RCAT Technology Details. 2011. Nov 30 2011. . 2. Dean, F .B., et al., Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Research, 2001. 11(6): p. 1095-1099. 3. Johne, R., et al., Rolling-circle amplification of viral DNA genomes using phi29 polymerase. Trends in microbiology, 2009. 17(5): p. 205-211. 4. Kingsmore, S.F. and D.D. Patel, Multiplexed protein profiling on antibody-based microarrays by rolling circle amplification. Current opinion in biotechnology, 2003. 14(1): p. 74-81. 5. Blanco, L., et al., Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. Journal of Biological Chemistry, 1989. 264(15): p. 8935. 6. GE Health. TempliPhi, Phi29 DNA Polymerase Based Rolling Circle Amplification of Templates for DNA Sequencing. 2011. Dec 4 2011. .

Wed Dec 07 23:57:41 2011 Li Yiding [email protected]

Dendreon

Product: Provenge Dendreon’s focus is utilizing active cellular immunotherapy (ACI) to combat aggressive metastatic cancers. The immune system is often able to detect and eliminate cells that undergo mutagenesis. This protective process also creates positive selective pressure that favors the proliferation of tumor cells able to alter their antigenic profile to avoid detection by the immune system. Thus, by assisting the patients’ own immune systems in recognizing tumor cells, Dendreon proposes a currently non-existent treatment system, Provenge, which leverages the innate capabilities of the immune system to treat prostate cancer and lower potential side effects associated with current treatment methods such as chemotherapy. Dendreon first identified a prostatic acid phosphatase (PAP) that is highly expressed by 95% of aggressive metastatic forms of prostate tumor cells. The PAP is joined to granulocyte macrophage colony stimulating factor (GM-CSF), which is a cytokine that acts as a leukocyte growth factor, to become the fusion protein PA2024. For the most effective treatment, patients need to have more aggressive form of prostate cancer that is well differentiated (Gleason score great than 7, range between 2-10, high score indicates more aggressive tumor) and are asymptomatic. To activate patients’ own immune system against prostate cancer, antigen presenting cells (APC) are isolated from patient blood samples via leukapheresis at weeks 0, 2 and 4. The blood is fed through a machine that separates the blood into its components via centrifugal forces, extracts the leukocytes, recombine plasma with red blood cells and platelets before finally reintroducing the sample back into the patient. APC is then separated from the leukocyte containing solution, cultured at Dendreon’s facility for 26-44 hours and then incubated with PA2024. After incubating APCs with PA2024, the APCs will uptake the fusion protein and present pieces of it on APC cellular membrane. The PA2024 presenting APCs are then infused back into the patients in the maximum possible amount from the preparation process. Upon re-entering the body, activated APC elicit patients’ cellular and humoral immune response, attacking prostate cancer cells that present PAP in sufficient quantities. Provenge is one of the next generation of cancer treatment methods that aims to harness the powerful immune system into fighting cancers. The method used by Dendreon is very creative, and results in a treatment that has decidedly less side effects and greater effectiveness. This is why I chose Dendreon.

Wed Dec 07 23:57:59 2011 Ahn Lisa [email protected]

Optogenetics

What is Optogenetics?: Optogenetics started out in 2005 at Stanford University in California with a team led by Karl Deisseroth. It is a fascinating technology based on gene therapy mediated introduction of light activated protein channels into cells. Two of such proteins that have been studied are channelrhodopsin-2 (chR) and halorhodopsin. The protein encoding sequences for these light sensitive channels are virally introduced into cells which then start to express the protein across its cell membrane. These genetically modified cells are then exposed to different light sources to produce either an excitatory or inhibitory effect. Stimulation of chR by blue light produces an excitatory response and stimulation of halorhodopsin by yellow light produces and inhibitory response. Why is this technology exciting?: This technology is exciting for those in the field of neuroscience. I am particularly interested in CNS plasticity and neuronal regeneration after a spinal cord injury resulting in paralysis. Currently, once damage is incurred in the level of the CNS, function can be lost without much hope for repair or regeneration of damaged neurons. For individuals with high-level spinal cord injury, loss of respiratory functions is a leading cause of death. Therefore many studies have explored ways to restore this lost function. The following is a study that utilized optogenetic technology to study reversal of motor loss resulting from spinal cord injury: Scientists transfected C2-C3 neurons(cervical level that controls the phrenic nerve that innervates the diaphragm) with sindbis virus to express the chR gene. Studies of C2-3 level spinal cord hemi-sected mice showed the ability of restoring normal breathing in mice using timed light impulses. In addition, chR expressing neurons were found in the diaphragm muscle and normal breathing ability was retained even after the light therapy was discontinued leading scientists to hypothesize that neuronal plasticity was facilitated through the optogenetic therapy. Optogenetics is a truly exciting technology that has very broad applications. All cells utilize ion channels for basic cellular function and therefore can be manipulated using specific light activated channel proteins to produce desired effects. Although, I have only briefly mentioned Optogenetics in pulmonary neuroscience, it has also been used to improve locomotion in spinal cord injured mice, improve bladder function in spinal cord injured mice, restore vision in blind mice lacking photoreceptor cells, stimulate cardiac muscle cells in-vivo to contract, and control behavior in animals. The rationale of choosing Optogenetics as an exciting new technology is precisely due to the potential for applications in functional regain after spinal cord injury- a disease once thought to have very little hope for recovery. In addition this technology is very exciting due to the broad applicability of Optogenetics across other specialties.