Crusio, Wim, E (1996) Gene targeting studies: New Methods, old problems. Trends in Neurosciences 19 186-187.

Gene targeting studies: New methods, old problems

Commentary by Wim E. Crusio

Génétique, Neurogénétique et Comportement

URA 1294 CNRS, Université de Paris V

45 rue des Saints-Pères

75270 Paris Cedex 06


Key words: gene targeting, neurobehavioral genetics, hippocampus, inbred mouse strains, neurological mutants, acetylcholine

Send correspondence to: Dr. Wim E. Crusio at the above address.

Tel. 19 33 1 42 86 22 06; Fax. 19 33 1 42 86 22 50; e-mail:

Techniques to create transgenic organisms or animals with targeted mutations ("knock-out" mutants) have become increasingly important tools in the neurosciences over the last few years. As always, new techniques, besides providing new tools to investigate problems or to test hypotheses, also give rise to unforeseen difficulties and it takes some time for researchers to become aware of this. Gene targeting techniques provide no exception and Gerlai's1 alert is very timely indeed. The complications described by Gerlai are, not very surprisingly, strongly reminiscent of those encountered in the study of spontaneous mutations in the mouse. In that field congenic strains have been used since many years2. Congenic strains are obtained by repeatedly backcrossing a mutant to an inbred strain. If we now consider the possible complications that may be encountered with this approach we may, in fact, distinguish two rather different types of "background" effects. As I will show in the following, the distinction between the two is crucial.

Hitchhiking donor genes

As noted by Gerlai, some residual donor genes that are closely linked to the mutated gene may still be present in a congenic line even after numerous generations of backcrossing. Of course, the presence of these contaminating hitchhiking genes may bias our experiments in a completely unpredictable direction, possibly leading to false positive or negative results. Up till now, no real solution to this problem was available, apart from continuing the backcrossing procedure over as many generations as feasible. Using modern techniques of gene transfer, Gerlai proposes several elegant possibilities by which researchers might control for confounding effects due to these kind of genes. I would like to suggest here an additional one.

In a way, targeted mutations are analogous to another widely used technique in the neurosciences, namely brain lesions. Knock-out experiments have in common with lesion experiments the assumption that the function of the lesioned structure equals the dysfunction of the residual organism. Also, in both types of studies we correlate certain effects with the presence or absence of an impaired structure (i.e., brain structure or gene). Obviously, correlational studies gain much in power if more than just two data points are available. For instance, we might exploit naturally occurring, non-pathological variations in neuroanatomy between individuals to uncover brain-behaviour relationships3. This approach has been named microphrenology by Lipp4 and may be augmented profitably by adopting a genetic strategy5. An analogous way to increase the number of data points in gene targeting studies might be the addition of transgenic animals. We could then compare the effects of underexpression (null mutants), normal expression (wild type), and overexpression (transgenics) of a certain gene. If different transgenic lines with different numbers of copies of the transgene are available, the number of data points can be increased even further. If this approach would render consistent results, we might be reasonably certain that the observed effects were due to the manipulated gene and not to genetic contamination by residual donor genes.

The recipient background

Second, there are the possible effects of the genetic background in its more classical sense of the recipient genotype (not necessarily inbred) to which the mutation has been introduced. In essence, we are here dealing with an interaction: the phenotypical effect of the mutation depends on the genetic background. A recent example of such a case was provided by the EGF knockout mice6,7, that showed very different phenotypes depending on the strain background upon which the EGF null mutant had been transferred. The latter result apparently surprised many, which in itself is quite amazing and telling because this should not have been necessary. Genotype-treatment interactions are a well-known phenomenon in the field of neurobehavioral genetics, be they differential expression of mutant genes depending on the genetic background8-10, or unequal effects of brain lesions11,12 or divergent effects of pharmacological treatments13 in different inbred strains.

Many researchers will probably consider such interactive effects a nuisance and, in consequence, choose to work with a genetically heterogeneous population. For a number of reasons, this would be a pity. First of all, although no interactive effects will be observed in such a population, this would be for the obvious reason that such interactions simply cannot be detected by such an experimental design. Of course, it is often argued that a heterogeneous population is more representative of the human population that it is supposed to model. What is overlooked is that such is only true if we are interested in human beings as a population, but not as individuals. If I were to suffer unpleasant secondary effects from some drug prescribed to me by my physician, it would be small consolation indeed to know that in the mean, this particular drugs has beneficial effects. To ignore an important experimental factor, in this case by using a genetically undefined population of experimental subjects, clearly is not an optimal research strategy.

Second, genotype-treatment interactions may provide the neuroscientist with a welcome additional tool. An instructive example has been provided by van Abeelen, who used a pharmacogenetic approach to investigate the role of acetylcholinergic (ACh) neurotransmission in the hippocampus in the regulation of mouse exploratory behaviour14,15. He used the inbred mouse strains C57BL/6 and DBA/2 that differ consistently in their levels of several exploratory acts in a novel environment: the former rates high, the latter low. Intrahippocampal injections with the anticholinergic drug methylscopolamine depressed the scores of the C57BL/6 strain, whereas those of the DBA/2 strain were enhanced. Equal treatments yielded opposite effects. In contrast, similar treatment with the acetylcholinesterase (AChE) inhibitor neostigmine depressed scores in both strains. Van Abeelen15 concluded that there exists a genotype-dependent cholinergic mechanism in the hippocampus that controls exploratory behaviour in mice. A functionally well-balanced ACh/AChE ratio appears to promote high exploration scores in C57BL/6 animals. Any injection with drugs that cause an imbalance in this ratio in either direction thus leads to a decline in exploratory activity. In DBA/2 mice a disequilibrium of this ratio (excess of ACh) leading to low levels of exploration was postulated. Correcting the imbalance by injecting anticholinergics then results in augmented exploration. Additional support for van Abeelen's hypothesis of hippocampal malfunction in DBA/2 mice has recently been provided16.

From the preceding, we may even hypothesize a probable differential expression of an ACh receptor null mutation: it might be expected to depress exploratory behaviour when expressed on a C57BL/6 background but to augment it on a DBA/2 background. If we should use, for instance, an F2 generation between these two strains as genetic background for our null mutation, then any conceivable result might be obtained, depending on the exact composition of our particular sample.

Concluding remark

As the above examples show, the study of genotype-treatment interactions and of naturally occurring interindividual variability may greatly enhance our understanding of the functioning of brain systems. A final note of caution may therefore be at his place here. Modern techniques like gene targeting and transgenesis provide exciting new opportunities for neuroscience research. However, in our excitement we should not forget that some questions might sometimes just as well, if not better, be addressed using less flashy and less fashionable techniques.


The preparation of this article was supported by the CNRS (URA 1294) and DRED (Université Paris V René Descartes).

Selected references

  1. Gerlai, R. (1996) Trends Neurosci. in press
  2. Snell, G.D. (1978) in Origins of Inbred Mice (Morse, H. C. III, ed), pp. 119-156, Academic Press
  3. Crusio, W.E., Schwegler, H. and Brust, I. (1993) Eur. J. Neurosci. 5, 1413-1420
  4. Lipp, H.P. et al. (1989) Experientia 45, 845-859
  5. Crusio, W.E. (1995) in Behavioural Brain Research in Naturalistic and Seminaturalistic Settings (Alleva, E. et al., eds.), NATO Advanced Study Institutes Series D, Behavioural and Social Sciences, Kluwer Academic Press
  6. Threadgill, D.W. et al. (1995) Science 269, 230-234
  7. Sibilia, M. and Wagner, E.F. (1995) Science 269, 234-238
  8. Ehrman, L. and Parsons, P.A. (1981) Behavior Genetics and Evolution, McGraw-Hill
  9. Fuller, J.L. and Thompson, W.R. (1978) Foundations of Behavior Genetics, C.V. Mosby
  10. Guastavino, J.M. and Cousin, X. (1992) Behav. Genet. 22, 724
  11. Ammassari-Teule, M., Fagioli, S. and Rossi-Arnaud, C. (1992) Physiol. Behav. 52, 505-510
  12. Donovick, P.J. et al. (1981) Physiol. Behav. 26, 495-507
  13. Broadhurst, P.L. (1978) Drugs and the Inheritance of Behavior. A Survey of Comparative Psychopharmacogenetics, Plenum
  14. van Abeelen, J.H.F., Ellenbroek, G.A. and Wigman, H.G.A.J. (1975) Psychopharmacologia 41, 111-112
  15. van Abeelen, J.H.F. (1989) Experientia 45, 839-845
  16. Paylor, R., Baskall, L. and Wehner, J.M. (1993) Psychobiology 21, 11-26