Gene targeting: technical confounds and potential solutions in behavioral brain research

Behavioural Brain Research 125 (2001) 13 – 21 www.elsevier.com/locate/bbr

Gene targeting: technical confounds and potential solutions in behavioral brain research Robert Gerlai * Lilly Research Laboratories, Lilly Corporate Center, Drop Code 0510, Indianapolis, IN 46285, USA Received 2 June 2000; accepted 25 January 2001

Abstract Gene targeting allows one to create null mutations in mice and to analyze how the mutant organism responds to the lack of a single gene product. This has facilitated the molecular dissection of such complex characteristics as mammalian brain function and behavior, including learning, memory, aggression, and maternal behavior to mention a few. However, the interpretation of the phenotypical changes that arise in null mutant mice has been questioned. The possibility that genes other than the targeted one may contribute to phenotypical alterations has been raised and the importance of compensatory mechanisms has been brought to attention. This review focuses on recent advances in the literature that illustrate the caveats associated with gene targeting and also presents an overview of potential solutions for the discussed problems. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Compensation; Gene targeting; Genetic background; Learning and memory; Linkage; Mouse; Strain differences

1. Introduction Five years ago Gerlai [22] raised several points based on elementary classical genetic considerations that questioned the interpretation of a large number of gene targeting studies. His debate article was followed by commentaries that confirmed and extended these arguments [7,10,33]. The problems pointed out in these papers did not prove that the findings of the cited gene targeting studies were wrong, but they clearly indicated that there was room for alternative interpretation of their data. Given the large amount of money and time invested in gene targeting experiments and given the popularity and touted advantages of the technique, the 1996 debate papers attracted much attention and induced a considerable debate regarding the utility of gene targeting [35]. Since then numerous suggestions for potential solutions have been discussed. The aim of this review is to summarize the problems and review some of the suggested solutions. * Tel.: +1-317-433-5244; fax: +1-317-276-7600. E-mail address: gerlai – [email protected] (R. Gerlai).

The problems can be divided into two main categories, both of which have general importance in gene targeting experiments concerned with brain function and behavior. The first is a cluster of problems associated with compensatory mechanisms. This problem is rather difficult and there is no general solution to avoid it. The second problem is associated with genetic background and linkage (the so called flanking region problem). Practical solutions to this problem will be presented. While the examples will be drawn mostly from the learning and memory field, the points illustrated are valid for any biological trait.

2. Compensatory mechanisms

2.1. The promise and the complexity With gene targeting one can knock out a gene in vivo and create a mutant organism that lacks the gene product. The promise of gene targeting has been to reveal the in vivo function of the gene of interest [2,25,26,38,49]. For example, Grant et al. [25] wrote: ‘…targeted disruption of genes provides a powerful tool

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for examining the role of specific proteins in the function of the brain’. However, the functional relevance of gene targeting has been questioned [39,44,45] because the mutation may lead to an avalanche of compensatory processes (up or down regulation of gene products) and resulting secondary phenotypical changes. Clearly, a null mutant organism may not only lack the product of a single gene but may also possess a number of developmental, physiological, or even behavioral processes that have been altered to compensate for the effect of the null mutation. Therefore, one may expect an array of complex phenotypical changes that may not be directly related to the function of the gene of interest.

2.2. The ‘helper’ genes Compensation may be due to genetic redundancy. Genetic redundancy in this context means that some putative ‘helper’ genes may be able to take over the function of the targeted one, e.g. become upregulated, and compensate for the absence of the targeted gene product. Crawley [7] explains that compensatory changes can mask the functional outcome of the mutation leading to an apparent absence of phenotypical effects of gene targeting. This has led some to conclude that the targeted mutagenesis technique is ‘wholly inappropriate for resolving the issues for which it was intended’ [45]. Others disagree with this view and argue that the ‘inability to observe a phenotypical change may not mean that there is no phenotypical change to observe’ [27]. The latter view is supported by the findings of Chen et al. [5]. These authors showed that although a null mutation in protein kinase Cg subtype (PKCg) in mice resulted in an apparently normal longterm depression (LTD) in the cerebellum of the mutants, LTD could be blocked by a PKC inhibitor only in control mice but not in the null mutants. This suggests that LTD was mediated, at least partly, by non-PKC dependent processes in the null mutant mice. The authors thus revealed a, yet unknown, alternative biochemical pathway that could compensate for the lack of PKCg and could support an apparently normal LTD in the mutant mice. Gene targeting, therefore, may enable the investigator to reveal novel biochemical pathways, functional interactions between the targeted gene and other genes.

2.3. Compensation for disrupted function ‘A’ leading to altered function in ‘B’ Importantly, a ‘compensatory’ change (a form of epistatic gene interaction) may not always lead to an absence of phenotypical change (false negative findings) but, on the contrary, may be the cause of observed phenotypical abnormalities. For example, assume gene

a serves hypothetical function ‘A’. Also assume that targeted gene a is compensated for by gene b which becomes upregulated in response to the absence of a gene product. The excess of gene b product is able to compensate for the lack of gene a product and no change is observed in function ‘A’ at the phenotypical level. However, overexpression of gene b product may have some pleiotropic effects, i.e. may affect functions other than ‘A’, similarly to the ways overexpression of genes alters brain function and behavior in transgenic mice [5,24]. These functional alterations when observed at the phenotypical level by the investigator will be assigned to gene a. Although such phenotypical alterations may be due to the introduced mutation, they need not reveal the function of the gene of interest per se because they may be related to it only indirectly. A skeptic could say that it is really impossible to assign specific functions to specific genes: genes act in concert and a disruption, let it be as targeted as one would like it to be, will always lead to a complex systemic response. Crusio [10] gives a simple, two component, example to show the interdependence of molecular components giving rise to complexity at the phenotypical level. He explains that pharmaco-genetic analyses of inbred strains of mice [52,53], C57BL/6 and DBA/2, led to the conclusion that hippocampal dependent exploratory behaviors rely on a delicate balance of acetylcholinesterase and acetylcholine levels. Therefore, the functional effect of genetic disruption of either component would depend upon the expression level of the other. Compensation, however, may not necessarily originate at the molecular level. Behavioral compensation translating into secondary molecular changes is also possible. Assume you have a mouse with genetically disrupted olfaction. This alteration may force the mouse to prefer visual stimuli to olfactory, which in turn may lead to multiple changes in neural processes and in brain areas involved in processing visual stimuli. An investigator then may conclude, incorrectly, that the targeted gene plays a crucial role in vision. Gene targeting studies trying to understand gene function in the organism may face similar enigmas. Teasing out the direct and indirect effects of the mutation is certainly not trivial and dissection of the molecular and neurobiological mechanisms underlying complex behavioral characteristics will require meticulous studies in which all possible affecting factors need to be controlled. Lathe [33] discusses several confounding factors to be taken into serious consideration in molecular genetic studies of brain and behavior. Instead of reiterating his arguments point by point I will summarize what I propose is the main issue: the necessity of using a ‘systemic approach’ in gene targeting [23].

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2.4. Biological organization is the important issue Although, the questions of genetic redundancy or compensatory mechanisms, or other factors confounding the effects of whatever artificial perturbation one applies to the living system are important, the principal problem is a systemic one that concerns biological organization and the functional units of this organization. From a geneticist’s viewpoint the units of biological organization are the genes and their function is to encode particular proteins. However, one may argue that when it comes to the question of phenotypical effects, genes may not be the units and the definition of their function may be more complicated. One may suggest that clusters of genes defined by higher organizational level phenomena, including developmental, physiological, or even behavioral, may represent the functionally relevant unit. Disruption of a single gene may alter a biochemical cascade within the functional gene cluster. Expression levels of the genes belonging to a functional cluster may change in concert. Investigation of such changes may allow the experimenter to reveal the biological organization of the brain. Perhaps the boundaries of these putative gene clusters are not sharp. Some genes may belong more, others less, to a specific functional gene group. This also implies that the gene group organization may not be orthogonal, i.e. some genes may belong to more than one functional group. Functional groups may be hierarchically organized. A smaller number of genes may define subgroups that may make up groups that in turn may be organized into super-groups, etc. Disrupting single genes will perturb the organism and will force it to respond in a way inherent to its biological organization. It is crucial to bear in mind, therefore, that the phenotypical changes one observes are the reflection of this particular organization. Instead of looking for the function of single genes, I propose that investigators should take a systemic organizational view into consideration. A conceptually similar approach, in which the effects of multiple system components are analyzed at the system properties level (see Metabolic Control Analysis in [30]), has gained acceptance in biochemistry [13,14].

2.5. Easier said than done Indeed, it is easy to outline what we should do. But, can we, and how? For example, how can one study the complex system and analyze the functionally relevant unit. There is no simple answer. A handbook written for the behavioral neuroscientist [11] discusses several potentially useful techniques one may want to employ to answer such questions. One could, for example, test gene expression changes triggered by the lack of the gene product in vitro or in vivo using quantitative RT-PCR or the recently developed gene-chip technology to see how genes respond in concert. One may also

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be able to alter, say knock out, the functional unit (cluster of genes) itself, instead of a single gene. An example for this latter approach, protein targeting with the use of immunoadhesins [4], has yielded valuable information on the involvement of a highly redundant cluster of protein tyrosine kinases, the EphA kinases [16,17,40], in cognition [18–21]. This approach is called protein targeting because this manipulation knocks out function at the level of the protein as opposed to the gene. The functional knock out is achieved at multiple relevant molecular targets and thus this approach also reduces the problems associated with compensatory mechanisms. Importantly, the compensatory changes potentially triggered by a genetic disruption will not only depend on the targeted gene itself and its involvement in certain molecular pathways but also on the background genotype. The potential confounding effects of background genes may represent a serious caveat in gene targeting. 3. Genetic background: an important confounding factor

3.1. Polymorphism in the genetic background may make the results of gene targeting studies difficult to interpret Consider the following example. Assume that targeted disruption of gene a leads to a differential expression of alleles b and B of gene b, and a regulatory change of gene b leads to different phenotypical effects depending on which allele (b or B) is present in the a null mutant organism. Consequently, polymorphism in the genetic background will not allow one to conclude with certainty that a particular phenotypical change observed in a null mutant animal was indeed due to the null mutation or to the genetic background. This issue is especially problematic if the genetic background of the null mutant animals is different from that of their wild type, control, counterparts. As discussed below, this is a definite problem in a large number of molecular neurobiology studies. To appreciate the problem, consider how gene targeting is performed.

3.2. Null mutant mice of gene targeting studies are often the F2 offspring of two mouse strains Most gene targeting [29] is carried out in cultured embryonic stem (ES) cells derived from the mouse strain ‘129’ (for a review see [22]). The 129-type ES cells carrying the targeted mutation are introduced into a blastocyst and the surviving chimeric embryos develop to term, are raised to adulthood, and are mated to ‘wild type’, i.e. non-mutated, mice. In case of successful germline transmission, these matings produce an offspring generation in which heterozygous null mutant

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mice are found. Problems arise, however, if the genetic background of the ES cell and of the mice to which the chimeras are mated are not identical. In several studies (for examples see [22]) the ES cells were derived from the mouse strain 129 but the chimeric mice were mated to, for instance, C57BL/6 (B6). The offspring of such matings (the F1 generation) therefore are not only heterozygous for the null mutant allele but have one set of chromosomes from strain 129 and another from B6 (see Fig. 1). These heterozygous mice, when sibmated, will produce a segregating F2 population in which, according to Mendel’s Law, homozygous null mutant, heterozygous null mutant and wild type mice are found.

3.3. Null mutant mice of hybrid origin are genetically different from their control littermates not only at the locus of the targeted gene but at other loci as well Comparison of the homozygous mutant, heterozygous mutant and wild type littermates of an F2

Fig. 1.

population appears to be an ideal way to reveal phenotypical changes brought about by the null mutation. Notably, however, such a segregating population constitutes mice with recombinant genotypes derived from the two parental mouse strains (see Fig. 1). The difficulties arising from this are threefold. First, the recombination pattern, i.e. which locus contains strain 129 and which B6 alleles, and whether in a homozygous or heterozygous form, may be different between littermates. This implies that not even wild type littermates of their mutant counterparts represent an appropriate control since their alleles could be different from those of the mutants not only at the locus of the gene of interest but also at other loci. This may lead to false

Fig. 1. Chromosomal constitution of mice generated by gene targeting. ES cells originating from mouse strain 129 carry one chromosome (gray) with the disrupted allele (white lesion) of the targeted gene. If these ES cells populate the germ-line in the chimeric mice, the mutation will be transmitted when the chimera is mated. A cross between a germline transmitting chimera and a C57BL/6 (B6) mouse (black chromosomes; panel A) will produce an F1 population (panel B) in which 50% of the animals will have one copy of the mutant allele (heterozygous mutants) and 50% of them will have no mutant allele (wild type animals) at the targeted locus. Using Southern blotting or PCR (Polymerase Chain Reaction) one can detect the presence of the mutant allele and identify the heterozygous mutant animals. If these animals are mated with each other, according to Mendel’s law, homozygous mutant (two mutant alleles), heterozygous mutant (one mutant and one wild type allele) and wild type (two wild type alleles) animals will be obtained. It is also important to remember, however, how genes at loci other than the targeted one will be inherited. Cross-over events during the meiotic process of gametogenesis will ‘shuffle’ the alleles of these background genes and will create recombinant chromosomes (panel C) which will characterize the genotype of the sperm and the egg of the F1 mice. The genotype of an F2 individual, therefore, will be represented by a pair of such recombinant chromosomes. For example, a homozygous null mutant mouse may have chromosomes a and b, a and c, or b and c; a heterozygous mouse may have one of the recombinant chromosomes with the lesion (a, b, or c) and another without the lesion (d, e, or f ); whereas a wild type control mouse may have chromosomes d and e, d and f, or e and f. Panel C shows that the null mutant allele of the targeted gene will be surrounded by 129-type genes, however, the wild type allele of the gene will be surrounded by B6 type genes. This linkage disequilibrium is simply due to the fact that the null mutant allele came from a strain 129 genetic background. In an animal produced from mating such F2 mice (F3 or the following generations), the null mutant allele could be surrounded by B6 genes only if, during the meiotic processes of gametogenesis, cross-overs occured precisely flanking both sides of the targeted gene, events whose combined probability is infinitesimally small. Panel D shows the probabilistic distribution of 129 (white) vs. B6 (black) alleles in an F2 segregating population. The depicted chromosomes thus represent the genotype ‘average’ of the F2 population. Note that in mice carrying the null mutation (chromosome ‘a’), the probability of finding 129 alleles on the mutant chromosome increases the closer a given locus is to the locus of the targeted gene. However, in mice carrying the wild type allele of the targeted gene (chromosome ‘b’), the probability of finding 129 alleles decreases the closer a given locus is to the locus of the targeted gene. Also note that as the distance increases from the locus of the targeted gene, the probability of the presence of 129 vs. B6 allele approaches 50 – 50%. (Modified from [22]).

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positive results. Second, due to the genetic variation resulting from the hybrid segregating background, detecting significant effects of the mutant gene of interest may be difficult yielding false negative results. These two problems can be alleviated by measuring larger number of animals, i.e. by increasing the power of statistical comparisons and decreasing the possibility of sampling error associated with recombination pattern differences between littermates. Increasing the sample size, however, will not solve the third problem which is associated with genetic linkage (see below).

3.4. The alleles of genes that surround the targeted locus will be of 129 -type in the null mutant mice and B6 in the wild type mice If the targeted mutagenesis is made in ES cells from strain 129, the chromosome with the targeted locus will carry alleles of genes of 129-type. The probability of genetic recombination is generally inversely related to the distance between the loci of the genes. Thus, the 129-type alleles of the genes whose loci are close to the locus of the mutated gene will remain together with the mutated allele of the gene of interest (see Fig. 1). In other words, any time the mutation is detected in a mouse, e.g. by Southern blotting, that particular animal will also carry the linked 129-type genes with high probability. Conversely, a non-mutant, control, animal most probably will not carry these 129-type alleles and will have B6 alleles instead if the 129-ES cell chimera was crossed to B6. In effect, the mutation can be seen as a marker for the 129-type genes linked to the locus of the targeted gene. Consequently, any phenotypical differences observed between mutant and control littermates of the hybrid genetic origin may be due either to the introduced null mutation or to the background genes linked to the targeted locus. Thus, one may find false positive results.

3.5. The beha6ioral alterations seen in null mutant mice may be due to the genetic background Empirical evidence supports that one may not be able to dismiss the genetic background problem or to argue that the potential effect of linked genes, and the effect of the genetic variation, in relation to the effect of the mutation is always negligible. For example, gene targeting studies investigating developmental consequences of the Epithelial Growth Factor (EGF) receptor disruption have demonstrated large background-genotype dependent null-mutation effects in mice [47,50]. The behavior genetics literature also provides ample evidence for large genetic differences between inbred mouse strains at the behavioral and neurobiological level [43,48]. This suggests that the

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potential effect of linked background genes cannot be ignored in gene targeting studies. Furthermore, strain comparisons have revealed that, unluckily, strain 129, which has been the choice of ES cell— gene targeting studies, is one of the most unique strains in terms of behavior, neuroanatomy, or neurophysiology [8,36,41,46,54]. For example, these animals are impaired in spatial learning tasks, a behavioral paradigm frequently used in molecular neurobiology studies [2,9,25,49,56] and they are considered passive [55] a behavioral trait that can be a confounding problem in several behavioral tests including the context dependent fear conditioning [2], in which freezing response is measured, the open field exploration test or tests of motor function such as the rotorod [3,6,31]. Moreover, strain 129 mice suffer from dysgenesis of the corpus callosum and possibly possess a number of other neuroanatomical peculiarities [36,54]. 129 mice also exhibit a peculiar ability to resist kainic acid induced excitotoxic cell death [46]. A further complication is that there are several substrains of 129 which are genetically distinct (Banburry conf) and thus depending on which substrain the ES came from, the contribution of 129 alleles to the phenotypical alterations seen in the null mice may be highly different. Indeed, in several gene targeting studies [2,3,6,42,49,56] in which the mice originated from a cross between a 129-ES cell chimera and another strain, the null mutant animals suffered from behavioral defects similar to those seen in the 129 strain from which the ES cell was derived (for review see [22]). It is therefore possible that the differences observed between mutant and control mice were in fact due to the genetic differences (in the linked background genes) between the inbred strains used in the generation of null mutant animals and not to the null mutation. Supporting this argument Kelly et al. [32] analyzed the D2 dopamine receptor-deficient (null mutant) mice and found that wild type strain 129 mice with unaltered functional D2 receptors were a virtual phenocopy of the predicted locomotor deficits ‘caused’ by the loss of D2 receptors in the null mutants. When they compared the wild type 129 and B6 parental strains, their F2 segregating generations, as well as a mutant (129× B6) segregating F2 generation, they found that genetic background contributed significantly to the phenotypical alteration seen in the null mutant mice. By studying mutant congenic strains (backcrossed to either B6 or 129 parental strains) they also showed a significant interaction between background genes and the targeted one, the former apparently exhibiting a greater effect on the behavioral phenotype [32]. Wolfer et al. [55] also provided evidence for similar background effects. On the other hand, Jia et al. [28] found, in another gene targeting study, that genetic background did not con-

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tribute to the observed electrophysiological alterations (elevated long-long term potentiation, LTP) in GluR2 null mutant mice. These authors analyzed both parental strains (CD1 and 129), their F2 hybrid population, and also a CD1×129 F2 population in which the null mutation was also segregating. Although reassuring to some, interestingly, Jia et al. could not detect a difference in LTP between 129 and CD1 mice, a finding discordant with the results of another group [1].

3.6. How could one a6oid the confounding effects of linked genes? A classical solution, one might suggest, would be to decrease the probability of contribution of background genes by backcrossing the mutant hybrid animals several times to the strain of choice, e.g. to B6, and create a congenic strain that carries the mutation on the desired genetic background. Backcrossing will undoubtedly increase the representation of the desired background genotype in the mutant animals. However, elimination of 129-type genes that surround the locus of the gene of interest would be a considerable undertaking: with 12 backcrosses (approximately 2 years of breeding) to B6, the length of the 129-type chromosome segment introduced to the B6 genome would be, on average, about 16 centiMorgans (cM) [15]. As the mouse genome covers about 1600 cM, this represents about 1% of the genome. Assuming the mouse genome contains 30,000 genes, then the introduced chromosome segment would be expected to have some 300 genes in it [15]. Because a potentially very large number of genes are involved in neural processes, the chance that some of the introduced linked 129-type alleles will have an influence on brain function and behavior is not negligible. Therefore, backcrossing is not the most optimal solution. Nevertheless, if no alternatives are available, it is recommended because it stabilizes the genetic background of the mutant line by reducing the variation in recombination patterns across generations. Zimmer [57] suggested a simple breeding scheme to test the contribution of background genes linked to the targeted locus. His suggestion was to breed a control wild type F2 generation (by crossing two wild type F1 mice) in addition to the mutant F2 generation (originating from crossing two mutant heterozygous F1 mice), where F1 is the hybrid between the 129 and B6 parental strains. Note that both mutant heterozygous and wild type F1 animals are obtainable after the founder chimeric (129 type sperm) mutant male is crossed to a B6 female. Zimmer then explains that if the behavioral alterations seen in the null mutants are due to the mutation, the mutant F2 generation should show segregation whereas the wild type F2 generation should not. Zimmer’s solution is correct in principle. However, it will most probably not work in practice, because the

behavioral traits are not qualitative, and the segregation characteristics of the F2 generations cannot be studied by simply counting the number of mice falling into three distinct categories (aa, ab, bb). Such a study would require complicated quantitative genetic analyses that need large sample sizes of subjects. Furthermore, Zimmer’s example also assumes that there is no interaction (epistasis) between the background genes and the targeted one and also that the behavioral trait in question is monogenic in the wild type F2 population, assumptions that are almost always not met in behavioral genetics research. Wolfer et al. ([12]; also see present issue) presented solutions to genetic background and the flanking gene problem, all based on different breeding schemes. First, they suggested to use an approach known as the ‘speed congenics’, which is essentially a backcross system that utilizes genetic markers to select those wild type recombinants that are most similar, in their recombination pattern, to the null mutant animals (see Fig. 1 for recombination patterns). Although useful in speeding up the process of making mutant and wild type recombinants more similar in their genetic background, this approach requires a lot of breeding and genetic testing, and will not produce a control mouse that is identical to the mutant at all loci but the targeted one. The second breeding scheme, ‘poor man’s choice’, [12], they suggested, does eliminate the necessity of genetic marker testing. It basically entails crossing recombinant heterozygous mutants back to wild type 129 and also to themselves (see present issue). These breedings will produce recombinant mice that possess the mutation at the target locus surrounded by 129 flanking genes, and mice with the wild type allele at the targeted locus also surrounded by129 alleles. The difficulty with the second scheme is that the two recombinants (mutant and wild type) are not identical, and, in fact, the wild type will have a larger 129 region flanking the target locus than the mutant. Thus, this breeding scheme is also unable to produce wild type and mutant mice with identical genetic backgrounds. Nevertheless, these breeding schemes are useful because they decrease the chance of false findings by reducing genetic differences in the background and also in the flanking region. To address the problems associated with using two inbred strains in the generation of null mutant mice several scientists gathered at a conference (Banbury Conference) and came up with well balanced critical recommendations that were published in a single paper [48]. The paper acknowledged the problems discussed by Gerlai before and also made several additional important points. For example: (1) ‘reports on genetic experiments must include a detailed description of the genetic background of the animals studied’; (2) ‘mutations must be maintained in congenic lines, and mutants must be analyzed in a defined hybrid (preferably

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F1) genetic background’; (3) proper wild type control mice may be generated ‘from crosses of F1 wild type mice in which the locus of interest derives from the genetic background of the ES cells. The identification of these mice requires isolation of polymorphisms within or near the targeted locus.’ The first suggestion may sound trivial but is often not followed, and thus is highly relevant. The second suggestion entails breeding the mutant mice with each of the two parental strains for repeated generations. Such breedings produce backcross populations in which the mutation is on a 129 strain or a C57BL/6 strain background in a heterozygous form. These heterozygous mutant congenic mice can then be crossed to generate a homozygous null mutant that is heterozygous (129 and C57BL/6) for all background loci except those that are linked closely to the targeted locus. The third suggestion is a very good one as, unlike the second, it allows the generation of wild type and null mutant mice with identical genetic background at all loci but the targeted one. Finally, the Banburry Conference also recommended that random insertional transgenic mice (mostly overexpressors of an inserted transgene) should be generated on the genetic backgrounds used for targeted mutagenesis, such as 129 and C57BL/6 in order to enable investigators to compare the effects of disruption or increased levels of gene expression. Additional suggestions have also been made. For example, rescue experiments are suggested to rule out the potential effects of linked 129-type genes [22]. In such an experiment one returns the missing functional protein by introducing a transgene that expresses the protein of the targeted gene or via a more direct systemic administration of the protein using osmotic minipumps for example. If such a manipulation restored the wild type phenotype in the null mutants then it would be a strong argument for the involvement of the targeted gene in the observed phenotypical alterations. Another potential solution is to generate ‘knock in’ mice in addition to the null mutant gene ‘knock out’ animals [22]. For instance, one could use homologous recombination to insert a small DNA marker flanking the gene of interest, without disrupting the gene. The knock in mice generated this way would have a fully functional targeted gene and they would have, on average, the same linked genes, and recombination pattern, as the knock out animals in which the gene of interest has actually been disrupted. Therefore, the knock in population would represent the ideal control, an idea that would work similarly to the third suggestion of the Banburry Conference [48] discussed above but without the necessity of existing genetic markers. Perhaps the most elegant solution to the background gene problem is to use inducible knock out techniques. The first studies with inducible tetracycline transactivator systems [37] or the cell type restricted CRE recombinase KO systems [51] to investigate brain function and

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behavior has shown the future to come. These techniques will allow the investigator to switch off or on the gene of interest at particular times and in particular places in the brain. Animals created with inducible systems will represent internal controls for the phenotypical analysis of mutations since the experimenter will be able to compare the pre and post induction phenotypes in the same individuals. However, as of today, these experiments also suffer from some complications associated with the lack of pure bred background [48]. Although the solutions suggested above are, or will soon be feasible, they are rather time consuming and expensive. A better, and more economical, solution is to avoid using hybrid mice all together. Generating null mutant mice with a pure genetic background could be done if one could cross the chimeras carrying the null mutation to a mouse of genetic background identical to that of the ES cell line used for the targeted mutagenesis. Unfortunately, this apparently simple solution is usually not considered, mostly for practical reasons. Paradoxically, the 129 strain from which ES cell lines are most easily derived is difficult to breed. Thus, while the choice of ES cell studies is still strain 129, adult strain 129 mice are often unwelcome in animal colonies. In addition, strain 129 mice are not preferred in animal behavioral studies either. It must be noted, however, that ES cell lines can be established from other strains of mice as well, including B6 [29]. Using a B6 cell line for gene targeting and crossing the created chimera to a B6 mouse would give rise to inbred B6 null mutant and wild type control mice in the F2 generation with no genetic polymorphism or confounding background genotype effects. Since B6 mice are often preferred in animal behavioral studies and are fairly easy to keep and breed, establishing B6 ES cell lines represents the optimal solution. It is unclear at this moment whether generating and using B6 ES cell lines is more problematic than working with 129 ES cells. Most scientists have preferred 129 ES cells. However, this may be due to historical reasons: the cell culture and harvesting techniques were developed for 129 ES cells because 129 strain of mice were most used for studying teratocarcinomas, a research line that eventually led to the development of ES cells (for examples see [29]). Nevertheless, a recent study has demonstrated that B6 ES cells can be generated and successfully used [34]. These ES cells enabled scientists to obtain a number of geneknock out lines with a success rate similar to that reported for ES cells derived from the 129 mouse strains [34].

4. Concluding remarks In summary, gene targeting in combination with thorough molecular, neurobiological, and behavioral

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examination of mutant animals it generates will provide us with detailed understanding of the molecular mechanisms underlying behavioral phenomena including learning and memory. Undoubtedly, behavioral and neurobiological traits are complex, often variable, and can be influenced by a large number of genes as well as environmental factors. In order to dissect such traits and to understand the interactions among the underlying biological mechanisms, it is crucial to control as many variables as possible. Understanding compensatory mechanisms and systemic responses to the absence of a gene product, and eliminating the confounding effects of background genes, are important steps forward that will facilitate our knowledge of how genes influence brain and behavior. Although controlling the genetic background is not always possible or feasible, the constant development of newer and newer recombinant DNA techniques, for example DNA microarrays or high resolution PCR techniques, together with the rapid accumulation of genetic knowledge such as the sequence of entire mammalian genomes will undoubtedly allow us to reach unprecedented levels of precision in our studies of the genetics of brain function and behavior.

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