Gene-targeting studies: new methods, old problems

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Gene-targeting studies of mammalian behavior

Behav. 48, 733-739 9 Smolen, A. et CJL (1986) PharmacoL Biochern. Behav. 24, 1077-1082 10 Church, A.C. and Feller, D. (1979) Phannacol. Biochem. Behav. 10, 335–338 11 Brase, D.A., Loh, H.H. and Way, E.L. (1977) J. Pharmacol. Exp. Ther. 201, 368-374 12 Miner, L.L. and Collins, A.C. (1989) PharrnacoZ.Biochem. Behav. 33, 469–475 13 de Fiebre,C.M. and Collins, A.C. (1993)]. Pharrrracol.Exp. Ther. 266, 1398-1406 14 Holsztynska,E.J., Weber, W.W. and Domino, E.F. (1991) Drug Metab. Dispos. 19, 48-53 15 Marks, M.J. et al. (1989) Pharrnacol. Biocherrr.Behav. 33, 679-689 16 Wainwright, P. and Deeks,S. (1984) Growth48, 192-197 17 Crusio, W.E. et al. (1990) Brain Res. 535, 343–346 18 Schopke, R. et aZ. (1991) Hippocanrpus1, 315-328 19 Livy, D.J. and Wahlsten, D. (1991) J Hered. 82, 459-464 20 Wahlsten, D. and Bulman-Fleming, B. (1994) Dev.Brain Res. 77, 203–214 21 Wahlsten, D. and Schalomon, P.M. (1994) Behav. Brain Res. 64, Acknowledgements 111-117 The author is 22 Schneider, R. et aL (1992) Behav. NeuralBioL 57, 198-204 indebted to the 23 Paylor, R. et al. (1994) Behav. Neurosci. 108, 810-817 many colleagues 24 Upchurch, M. and Wehner, J.M. (1988) Pharrnacol. Biochem. Behav. 29, 325–329 who have 25 Upchurch, M. and Wehner, J.M. (1989) Behav. iVeurosci.103, contributedto this 1251–1258 discussion. 26 Morris, R.G.M., Garrud, P. and Rawlins,J.N.P. (1982) Nature 297, 681-683 J.J. Mullins, 27 Upchurch, M. and Wehner, J.M. (1988) Behav. Genet. 18, S. Pncsinerand 55-68 J Wehnerare 28 Hogan, B., Lacy, E. and Costantini, F. (1986) Manipulatingthe MouseEmbryo, Cold SpringHarborLaboratoryPress thanked for 29 Gerlai, R. (1995) TrendsNeurosci. 19, 177-181 communicating 30 Carvallo, D., Canard, G. and Tucker, D. in TransgenicAnimak, Generation and Use (Houdebine,L.M., cd.), HarwoodAcademic unpublisheddata.

(in press) 31 Allen, N.D., Norns, M.L. and Surani, M.A. (1990) Cell 61, 853-861 32 Deng, C. and Capecchi, M.R. (1992) A40Z.Cell. BioL 12, 3365-3371 33 te Riele, H., Maandag, E.R. and Bems, A. (1992) Proc. NatZ Acad. Sci. USA89, 5128–5132 34 Collinge,J. et aZ.(1994) Nature 370, 295-297 35 Umemori, H. et aL (1994) Nature 367, 572-576 36 Li, Y. et al. (1994) Cell 76, 427-437 37 Skarnes, W.C., Auerbach, B.A. and Joyner, A.L. (1992) Genes Dev. 6, 903-918 38 Threadgill, D.W. et al. (1995) Science 269, 230-234 39 Ledermann, B. and Biirki, K. (1991) Exp. Cell Res. 197, 254-258 40 Kawase,E. et al. (1994) Int. J. Dev. BioL 38, 385-390 41 Yagi, T. et aL (1993) AnaL Biochenr.214, 70-76 42 Winson, J. (1978) Science 201, 160-163 43 Stewart, M. and Fox, S.E. (1990) Trends Neurosci. 13, 163-168 44 Juraska, J.M. (1991) Psychoneuroendocrinology16, 1–3 45 Jacobs, L.F. et aZ. (1990) Proc. Natl Acad. Sci. USA 87, 6349–6352 46 McEwen, B.S. (1992) Neuroendocrinology37(Suppl. 3), 1-10 47 Fordyce, D.E. and Wehner, J.M. (1993) Brain Res. 619, 111-119 48 Morelli de Liberti, S.A. et al. (1985) }. Neurochem. 45, 1055–1061 49 Prechel, M.M., Audhya, T.K. and Simmons, W.H. (1989) ]. Pineal Res. 6, 1-7 50 Peinado, M.A. et al. (1990) J. Neural. Trarrsm.81, 63-72 51 Grant, S.G.N.et al. (1995) Genes Dev. 9, 1909-1923 52 Lathe, R. and Morris, R.G.M. (1994) NeuropathoI. Appl. Neurobiol. 20, 350-358 53 Gossen, M. and Bujard, H. (1992) Proc. NatI Acad. Sci. USA89, 5547-5551 54 Kuhn, R. et al. (1995) Science269, 1427-1429 55 Dobie, K.W. et al. Proc. Natl Acad. Sci. USA(in press)

Gene-targeting studies: new methods, old problems Trends Neurosci. (1996) 19, 18&187

Commenta~ by Wim E. Cru.sio G&%que, Neurogt%&ique et Comportment, URA1294 CNRS, Universityde Paris V,45 rue des Saints-Pires,75270 Paris Cedex06, France.

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these contaminating hitchhiking genesmight bias our experiments in a completely unpredictabledirection, possibly leading to false positive or negative results. Until 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 the confounding effects of these genes. I wouldlike to suggestan additional one. In a way, targeted mutations are analogous to another widely used technique in the neuroscience, namely brain lesions. Knockout experiments and lesion experiments have in common the assumption that the original function of the lesioned structure is revealedby the dysfunction of the residualorganism. Also, in both types of studies we correlate certain effects with the presence or absence of an impaired structure (that is, brain structure or gene). Obviously, correlational studiesgain much in powerif more than just two data points are available. For example, we Hitchhikingdonor genes might exploit naturally occurring, non-pathological First, as noted by Gerlai, some residualdonor genes variations in neuroanatomy between individuals to that are closely linked to the mutated gene might still uncoverbrain–behaviourrelationships3.This approach be present in a congenic line even after numerous has been named microphrenologyby Lipp4and might generationsof backcrossing.Of course,the presenceof be augmentedprofitablyby adoptinga geneticstrate~.

Techniques to create transgenic organismsor animals with targeted mutations (’knockout’ mutants) have become increasingly important tools in the neuroscience over the past 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 provideno exception and Gerlai’slalert 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 for many yearsz.Congenic strains are obtained by repeatedlybackcrossing a mutant to an inbred strain. If we now consider the possiblecomplications that might be encounteredwith this approach we might, in fact, distinguish two rather different types of ‘background’effects. AsI will show in the following,the distinction between the two is crucial.

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Gene-targeting studies of mammalian behavior

An analogous way to increase the number of data points in gene-targetingstudiesmight be the addition of transgenic animals. We could then compare the effects of underexpression(null mutants), normal expression (wildtype), 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 increasedeven further. If this approach were to 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.

those of the DBA12strain wereenhanced. Equal treatments yielded opposite effects. In contrast, similar treatment with the acetylcholinesterase (AChE) inhibitor neostigminedepressedscoresin both strains. It was concluded by van Abeelen15that there exists a genotype-dependentACh-activatedmechanism in the hippocampusthat controls exploratory behaviour in mice. A functionally well-balancedAChlAChE ratio appears to promote high exploration scores in C57BL/6animals. Any injection with drugsthat 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. The recipientbackground Correctingthe imbalanceby injecting ACh antagonists Second, there are the possible effects of the genetic then results in augmented exploration. Additional backgroundin its more classical sense of the recipient support for van Abeelen’s hypothesis of hippocamgenotype (not necessarily inbred) to which the mu- pal malfunction in DBA/2mice has recently been tation has been introduced. In essence, we are here provided”. From this, we might even hypothesize a probable dealingwith an interaction: the phenotypicaleffect of the mutation depends on the genetic background. A differential expression of an ACh-receptor null murecent example of such a case wasprovidedby the epi- tation: it might be expected to depress exploratory dermal growth factor (EGF)-knockout miceb”, that behaviour when present on a C57BL16 background, showed very different phenotypes depending on the but to augment it when present on a DBA12backstrain background upon which the EGF null mutant ground. If we should use, for example, an F2 generhad been transferred.This result apparentlysurprised ation between these two strains as the genetic backmany, which in itself is quite amazing and telling ground for our null mutation, then any conceivable because it should not have been necessary.Genotype- result might be obtained, depending on the exact treatment interactions are a well-knownphenomenon compositionof ourparticularsample.Thisis an obvious in the field of neurobehaviouralgenetics, be they dif- drawbackof using such a genetically heterogeneous ferential expression of mutant genes depending on population. the genetic background&lO,unequal effects of brain Concludingremarks lesions1112,or divergent effects of pharmacological Asthe aboveexamplesshow, the studyof genotypetreatments13in different inbred strains. Many researcherswill probablyconsider such inter- treatment interactions and of naturallyoccurringvariactive effects a nuisance and, in consequence, choose ability betweenindividualsmight greatlyenhance our to work with a genetically heterogeneouspopulation. understandingof the functioning of brain systems. A For a number of reasons, this wouldbe a pity. First of final note of caution might therefore be apt here. all, although no interactive effects will be observedin Modern techniques like gene targeting and transgensuch a population, this would be for the obvious esis provide exciting new opportunities for neuroreason that such interactions simply cannot be science research. However, in our excitement we detectedby such an experimentaldesign. Of course,it should not forget that some questions sometimes is often argued that a heterogeneous population is might be just as well, if not better, addressedusing more representative of the human population that less-flashyand less-fashionabletechniques. it is supposed to model. What is overlooked is that such is only true if we are interestedin human beings Selectedreferences 1 Gerlai, R. (1996) TrendsNeurosci. 19, 177-181 as a population, but not as individuals. If I were to suffer unpleasant secondary effects from some drug 2 Snell,G.D. (1978) in OriginsofInbred Mice (Morse,H.C., III,cd.), pp. 119–156, AcademicPress prescribedto me by my physician, it would be small 3 Crusio, W.E., Schwegler, H. and Brust, L (1993) Eur. J Neurosci.5, 1413-1420 consolation indeed to know that in the mean, this 4 Lipp, H.P. et aL (1989) Experiential45, 845–859 particular drug has beneficial effects. To ignore an 5 Crusio, W.E. (1995) in BehaviouraZBrain Research in Naturalistic important experimental factor, in this case by using and Semir@uralistic Se~’ngs (Alleva,E. et aL, eds), pp. 323–342, a genetically undefined population of experimental KluwerAcademicPress 6 Threadgill, D.W. et al. (1995) Science 269,230-234 subjects, clearly is not an optimal research strategy. and Wagner, E.F. (1995) Science 269, 234-238 Furthermore,genotype-treatmentinteractionsmight 87 Sibilia,M. Ehrrnan, L. and Parsons, P.A. (1981) Behavior Genetics and providethe neuroscientist with a welcome additional Evolution,McGraw-Hill 9 Fuller,.J.L.and Thompson, W.R. (1978) Foundations ofBehavior tool. An instructive example has been provided by Genetics, C.V. Mosby van Abeelen, who used a pharmacogenetic approach 10 Guastavino, J.M. and Cousin, X. (1992) Behav. Genet. 22, to investigatethe role of AChneurotransmissionin the 724 hippocampus in the regulation of mouse exploratory 11 Ammassari-Teule,M., Fagioli, S. and Rossi-Amaud,C. (1992) PhysioZ.Behav. 52, 505-510 behaviour14’15.He used the inbred mouse strains 12 Donovick, P.J. et al. (1981) Physiol. Belrav. 26, 495-507 C57BL/6and DBA/2that differ consistently in their 13 Broadhurst, P.L. (1978) Drugs and the Inheritance of Behavior. levels of several exploratory acts in a novel environA Surveyof ComparativePsychophannacogenetics,Plenum ment: the C57BL/6 strain rates high whereas the 14 van Abeelen,J.H.F., Ellenbroek, G.A. and Wigman, H.G.A.J. (1975) PsychophamnacoZogia41, 111-112 DBA/2strain rates low. Intrahippocampal injections 15 van Abeelen,J.H.F. (1989) Experiential45, 839-845 with the ACh antagonist methylscopolamine 16 Paylor, R., Baskall, L. and Wehner, J.M. (1993) Psychobiology 21, 11-26 depressedthe scores of the C57BL16 strain, whereas TINS Vol. 19, No. 5, 1996

Acknowledgements Thepreparationof this a~”clewas supportedby the CNRS(VRA1294) and DRED (Lhriversittl Paris V Ren6Descartes).

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