The knockout strategy enables the generation of mutant mice lacking a 

specific gene and, therefore, the corresponding protein20 (see Fig. 1). This 

technology relies on the fact that blastocyst-derived embryonic stem (ES ) 

cells can be grown in vitro and modified by transfection. In particular, it is 

possible to substitute the wild type copy of a given gene by either a non 

functional copy of this gene or a gene encoding a protein with altered 

properties. In the example shown in figure 1, a portion of the gene is 

substituted by a sequence encoding resistance to the antibiotic neomycin 

(neo) which is used to select the embryonic stem cells that have undergone 

recombination. These engineered ES cells are injected into a recipient 

blastocyst and contribute to the formation of a chimeric mouse. Some of the 

chimeras will transmit the mutation to their germ line. The resulting 

heterozygous mice are bread to generate mice that are homozygous for the 

desired mutation. The ability to generate such mutant mice brings into the 

mammalian field the considerable power of direct genetic manipulation, 

which was until recently limited to simpler organisms such as bacteria, yeast 

or Drosophila. Mutant mice have been generated that lack the 5-HT1B21 and 

the 5-HT2C22 receptors.<P>

	The 5-HT1B mutant mice did not exhibit any obvious developmental or 

behavioral defects. However, the hyperlocomotor effect of the 5-HT1A/1B 

agonist RU24969 was absent in the mutant mice indicating that this effect is 

mediated by the 5-HT1B receptors. Moreover, in the isolation induced 

agression test the mutant mice were more aggressive than the wild types 

mice. When confronted with a male intruder, mutant male mice that had been 

isolated for one month attacked the intruder faster and more intensely than 

did wild type mice. These results together with the finding that certains 5-

HT1 agonists termed ÓserenicsÓ have anti-aggressive properties suggest an 

involvement of 5-HT1B receptors in the modulation of aggressive behavior.

<P>	Mice lacking 5-HT2C receptors are overweight as a result of abnormal 

control of feeding behavior. This result is in good agreement with the fact that 

nonselective 5-HT2C receptor agonists such as mCPP have been shown to 

suppress appetite. these mice are also prone to spontaneous death from 

seizures. In addition, the mutant mice displayed a lowered threshold for 

metrazol-induced seizures. Interstingly, this effect could be mimicked in 

wild-type animals with a non-selective 5-HT2C receptor antagonist. This 

result supports the hypothesis that the epileptic phenotype does not arise 

from an abnormal brain development and reflects a normal adult function of 

the 5-HT2C receptor.

<P>	The advantage of the knockout strategy is that the block of expression is 

total and highly specific. However the consequences of such mutations are 

exerted throughout the development of the mouse. Therefore if the protein of 

interest plays a developmental role that might be different from its role in 

the adult, the mutant might exhibit developmental abnormalies that might 

preclude the analysis of the adult phenotype. The second potential difficulty 

with the knockout strategy is that  of the nervous system is plastic and might 

be able to compensate for the missing protein by either rewiring certain brain 

areas or by regulating the expression of the other proteins that participate in 

the same functions as the mutated protein. To circumvent the problem of 

compensatory changes it may be advantageous to be able to delay the 

knockout of a particular receptor until the mutant animal becomes an adult 

<P>
However the classic constitutive knockout might reveal unexpected 

developmental roles for particular receptor. In addition such constitutive 

knockouts might represent a good model for genetic deseases. For example 

recently generated mice that lack a functional gene for the enzyme Mono 

Amine Oxidase type A (MAOA) might represent an animal model for a 

mutation which has also been found in the human population ( ). 

Interestingly both mice and man lacking this enzyme display increased 

aggressive behavior.

<p>
<img src="ko.gif">