Class 15
Audio from 2006 (WMA)

IN VITRO & TISSUE CULTURE ASSAYS OF PROTEIN AND GENE FUNCTION

Protein from cloned genes;

cell hosts; affinity labels; promoters

Class 16
Audio (WMA)

Dale and Schantz: 224-225, 241-244

Strachan and Read: 545-558

Class 17

Audio (WMA)

protein-protein interaction;

mutational mapping of promoter-enhancers

Clonal selection and in vivo quantitation of protein interactions
with protein-fragment complementation assays, I. Remy and S.W. Michnick
PNAS 96, 394–5399, 1999

Class 18

Audio (WMA)

Protein-DNA interactions

DNA microarrays

High throughput DNA sequencing

Class 19
No Audio: Sorry, recording failed.

High throughput DNA sequencing, cont.

CHIP-Chip

Proteomics

MAMMALIAN CELL GENETICS:

Cultured mammalian cells as microorganisms; cell mutants

Animal cell culture:  Lodish (online chapter)

Purine and TMP Metabolism:  Biosynthetic pathways

Legend to biosynthetic pathways figure.

Class 21
Audio (WMA)

MAMMALIAN CELL GENETICS cont.:  mutants cont.; cell fusion, selective medium, transfection;  gene amplification, co-amplification

Reff, MR, Barnett, RS, and McLachlan, KR. US Patent 6,413,777. Method for integrating genes at specific sites in mammalian cells via homologous recombination and vectors for accomplishing the same. (skim, but understand the strategy and the vectors)
Florian Wurm  Nature Biotechnology  22, 1393 - 1398 (2004) Production of recombinant protein therapeutics in cultivated mammalian cells

Class 22
Audio (WMA)

ACHIEVING HIGH LEVEL RECOMBINANT PROTEIN PRODUCTION IN MAMMALIAN CELLS

maximizing gene expression, controlling chromosomal position

IMMUNOGLOBULIN STRUCTURE AND FUNCTION antibody-dependent cellular cytotoxicity

Monoclonal antibodies:

Strachan and Read: 613-616

In Janeway et. al., Immunobiology (online): A-12. Monoclonal antibodies.

Class 23

Audio (WMA)
(missed 1st ~15 min)
Audio (mp3)
Full lec, lesser quality
(compliments of Yasser)

MONOCLONAL ANTIBODIES AS DRUGS:

hybridomas

SECOND GENERATION THERAPEUTIC PROTEINS I:

Manipulating protein glycosylation

Glycosylation:  Stryer (online): Glycoproteins (Sec.11.3 incl. figs.)

Naoko Yamane-Ohnuki, Satoko Kinoshita, Miho Inoue-Urakubo, Machi Kusunoki, Shigeru Iida, Ryosuke Nakano, Masako Wakitani, Rinpei Niwa, Mikiko Sakurada, Kazuhisa Uchida, Kenya Shitara, Mitsuo Satoh.  Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.   Biotechnol Bioeng. 2004 Sep 5;87(5):614-22

Class 24
Audio (WMA)

SECOND GENERATION THERAPEUTIC PROTEINS II: selection for improvements on nature

Mutagenic manipulation:

GC Rice, DV Goeddel, G Cachianes, J Woronicz, EY Chen, SR Williams, and DW Leung. Random PCR mutagenesis screening of secreted proteins by direct expression in mammalian cells. PNAS 1992; 89:5467-5471

Class 25
Audio (WMA)
(missed last ~10 min)
Audio (mp3)
Full lec, lesser quality
(compliments of Yasser)

ENZYME-DIRECTED EVOLUTION and CREATION of NEW PROTEINS:

DNA shuffling, enzyme evolution in vitro

DNA shuffling:
Maxygen Website: Publications on DNA shuffling. OPTIONAL

RNA-TARGET THERAPY:

Antisense RNA, RNAi, correction of splicing defects

Class 27
Audio (WMA)

APTAMERS AND RIBOZYMES AS THERAPEUTICS AND BIOSENSORS:

Selection of oligomers that bind a target, aptamers as alternatives to MAbs 

C Frauendorf, A Jaschke. Detection of small organic analytes by fluorescing molecular switches. Bioorg Med Chem (2001) 9(10):2521-4.

517 Hamilton

FINAL EXAM

Focused directly on material of the latter half of course, but assuming knowledge of the first half.