Knockout And Transgenic Strategies

The development of targeted gene disruption has been one of the more important breakthroughs in unraveling the molecular underpinnings of behavior. The aim is to selectively inactivate a gene of interest (i.e., disrupt a targeted gene) and to compare this so-called knockout animal (usually a mouse) with a control or wildtype animal that has all its genes intact. Observed differences can then be attributed to the gene in question. Hence, by comparing the behavior and underlying neuronal processes of knockouts and wild types, one can deduce the function of the gene and determine its effects on complex traits.

It is beyond the scope of this article to discuss the technical details of this technique. Briefly, this technique relies on the fact that embryonic stem cells can be grown in vitro and modified by transfection. By homologous recombination the wild-type (or normal) gene in question can be replaced by a disrupted form of the gene that for example, codes for an antibiotic. The latter is used to select for recombined embryonic stem cells. These engineered cells are then injected into a recipient embryo to form chimeric mice (mice that have both cells with the normal gene and cells with the disrupted gene). Next, only those mice that have transmitted the mutation in their germline are used to generate nonchimeric mice carrying this artificial mutation.

To date more than 30 genes that influence male offensive behavior have been identified using the knockout technique (Table II). Interestingly, the involvement of the MAOA gene in abnormal social behavior (including aggression) was confirmed by a knockout study. Mice having a disrupted MAOA gene show more aggressive behavior than control mice. Also, the relatively nonspecific consequences of this gene defect were confirmed: MAOA-deficient mice tremble, have difficulty in righting themselves, and are fearful and blind. Other knockout lines show a more specific aggressive behavioral profile. For instance, mutant mice lacking the 5-HT1B receptor do not exhibit any obvious developmental or behavioral defects but do show enhanced aggressive behavior. In this respect, it is notable that about half of all studies on gene knockouts and aggression indicate that there is either a direct or an indirect relation between aggression and serotonin. Although the exact mechanisms through which serotonin exercises its influence on aggressive behavior is far from clear, data suggest that a low serotoninergic transmission is associated with increased aggressive behavior.

At this point, some comments on knockout studies must be made. First, there is always the possibility that the knockout and the wild type differ in more than one gene. This so-called "flanking gene'' problem results from the technical procedure per se and may lead to false positives. A second problem is the genetic background of the knockout, which is either randomized or, at best, homogeneous. In the latter case, the obtained knockout is repeatedly crossed back to mice from the same inbred strain. After many predefined backcrosses, usually 10 or more, in which the presence of the mutated allele is checked every generation, the background is said to be homogeneous. A comparison between the knockout and the inbred strain will then yield information on the effect of the knocked out gene on a specific genetic background. However, it is certainly possible that an inactivated gene

Table II

Genes or Gene Variants Found to Affect Male Offensive Aggression in Knockout Studies

System

Gene

Symbol

Effect0

Steroid

Aromatase

Cyp19

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