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Published December 11, 2014 Targeted Mutagenesis of Mouse Developmental Genes Kirk R. Thomas Howard Hughes Medical Institute, University of Utah, Salt Lake City 84112 ABSTRACT Targeted mutagenesis of genes in mouse embryo-derived stem ( E S ) cells provides a means t o systematically alter the genome of a mammalian organism. This technology permits the in vivo mutagenesis of genes whose role in development has heretofore been merely the subject of speculation. Gene targeting in ES cells is mediated via a cellular homologous recombination machinery. Parameters that optimize the detection of homologous recombinants have been developed by targeting mutations t o the Hprt gene. These gene-targeting protocols have been used to demonstrate the requirement in mouse development of one gene, the proto-oncogene Wnt-1. Mice homozygous for null mutations in Wnt-1 show severe abnormalities in the cerebellum and midbrain and generally, but not always, die shortly after birth. Key Words: Mice, Gene Targeting, Embryo-Derived Stem Cells, Embryonic Development J. Anim. Sci. 1994. 72(Suppl. 3):1-8 A typical mammalian cell contains approximately of the expression of this complex genome is largely responsible for the ordered progression seen during the development of a single fertilized egg to an adult organism containing perhaps 1014 cells. The elucidation of the specific developmental roles for each of these individual genes can come from several experimental sources. For example, studies of transcriptional or translational expression patterns lead to inference of the position of specific genes in the developmental pathway. Alternatively, the role of a given gene may be defined by analyzing the development of animals defective in that gene. The characterization of mutant phenotypes not only exposes the temporal and spatial position of a gene’s action, but may lead to a functional understanding of the gene product a t the biochemical level. Traditionally, developmentally important genes have been characterized by the recognition of an abnormal phenotype followed by identification of the gene and the mutation responsible for that phenotype. Although this approach has uncovered a number of important processes in organisms as varied as bacteria and humans, its utility generally decreases with the increasing complexity of the organism to be studied. In the case of mammals, for example, limitations imposed by generation time, mutation frequency, diploidy, and internal embryonic development have severely limited the number and type of spontaneously arising developmental mutations. Thus, among the nearly 1,000 known mice mutants, the class of recessive, embryonic lethal mutations is severely underrepresented (Lyon and Searle, 1989). Recent advances in cellular and molecular biology, however, now permit the investigator to avoid the limitations imposed by life history and generate predesigned mutations in a model experimental mammal, the mouse. Although the prospect of systematically mutating each of the 100,000 mouse genes and then assessing the developmental consequences of these mutations is quite daunting, a short-list of candidate developmental genes can be constructed. Such genes may include those encoding growth factors, proto-oncogenes, or genes whose homologues are known to function developmentally in other organisms. Because a large number of these potential “developmental genes” now exist as cloned laboratory reagents, they can be mutated in vitro, and targeted back to the host organism (Capecchi, 1989). This paper will include a discussion of the technology that now permits the modification of virtually any mouse gene and will discuss a specific example of a targeted mutation that alters the normal murine developmental pathway. lo5 genes. The precise regulation Mutagenesis by Gene Targeting The targeted mutagenesis of a gene is a multi-step process. A copy of the gene of interest is first cloned and mutated in vitro and is then introduced as a “targeting vector“ into the host cell. Once inside the cell, the mutagenicity of the targeting vector depends on the homologous recombination machinery of its host. This machinery, undoubtedly a component of a DNA-repair pathway, can catalyze the exchange of genetic information between two homologous pieces of DNA. If this exchange involves the mutated sequence and its chromosomal, wild-type homologue, a genetic alteration can be fixed in the genome. 1 2 THOMAS An extremely efficient homologous recombination machinery exists in a variety of single-cell organisms, such that the precise genomic modification of simple eukaryotes such as fungi and certain protozoa has become routine (Hinnen et al., 1978; Losanne and Spudich, 1987; Cmz and Beverley, 1990; Ten-Asbroek et al., 1990). The genome of higher eukaryotes, however, has not been as tractable to manipulation. One reason for this is the relative inefficiency of the mammalian homologous recombination machinery. A targeting vector introduced into a cultured mammalian cell, for example, has a very low chance of interacting with its endogenous homologue. Rather, exogenous DNA is integrated into the host genome at apparently random (i .e., sequence-independent) chromosomal locations. The non-homologous reaction between exogenous and endogenous DNAs can be from lo2 to lo4 more frequent than the homologous interaction (Thomas et al., 1986; Thomas and Capecchi, 1987). An additional obstacle to a useful mutagenesis protocol is that the desired modification should ideally be delivered to the germ line of the host organism. In the case of the mouse, there are a limited number of options for such delivery. One method takes advantage of viral vectors to introduce DNA to early-stage embryos (Jaenisch, 1976). Similarly, DNA injected directly into the pronuclei of newly fertilized eggs will be stably incorporated into the genome and thus populate all derivative cells in the ensuing adult. The low frequency of homologous recombination limits the use of these methods and, in fact, only a single instance of homologous recombination has been reported following the injection of DNA into a fertilized mouse egg (Brinster et al., 1989). To obtain this single recombinant, 10,000 eggs were injected and implanted and nearly 2,000 progeny were then analyzed at the DNA level. An alternative strategy is to introduce the targeting vector into embryo-derived stem ( ES) cells (Thomas and Capecchi, 1987; Capecchi, 1989). Although these cells also exhibit low recombination frequencies, their use reduces the screening of recombinants from the animal to the cell culture level. These cells, derived from pre-implantation-stage mouse blastocysts, can be grown and manipulated in culture (Bradley et al., 1984; Gossler et al., 1986; Robertson et al., 1986). Following their transformation by exogenous DNA, these cells can be screened and the appropriate recombinants cloned and transferred to another blastocyst. The ES cells can then colonize the host embryo and their progenitors can contribute to all cell types in the developing mouse, including germ cells. Thus, a mutation generated in vitro can be propagated indefinitely. For all practical purposes, gene targeting in mouse ES cells is the only currently available route for predetermined modification of endogenous genes in a mammalian genome. Optimization of Gene Targeting Although ES cells clearly contain the capacity t o mediate homologous recombination, the low frequency of this recombination creates a logistical problem in isolating recombinants from a pool containing nontransformed cells and non-homologous recombinants. Two basic approaches have been explored to overcome this obstacle. One is to increase the ratio of homologous vs non-homologous recombinants; the other is to specifically enrich for the growth of the homologous recombinants. The optimization of the experimental parameters used for gene targeting in ES cells has been largely done by mutating the Hprt gene (Thomas and Capecchi, 1987; Doetschman et al., 1988; Deng and Capecchi, 1992; Thomas et al., 1992; Deng et al., 1993). This gene, encoding the enzyme hypoxanthine phosphoribosyltransferase, has a number of qualities making it the model gene. First, the gene is not required for cellular viability. Second, cells carrying mutations in Hprt are resistant to the purine analogue 6 thioguanine ( 6-TG), and can thus be selected for in culture. Finally, the Hprt gene resides on the Xchromosome, such that in male-derived cells, only a single mutational event is required to render a cell hprt- (and thus 6-TW). The most commonly used gene targeting strategy is the replacement of sequences at the target locus with sequences from the targeting vector. A typical gene replacement at the Hprt locus is illustrated in Figure 1. The vector contains sequences homologous to the target gene that have been mutated by the presence of a selectable genetic marker (in this case the neor gene from E. coli) inserted within the protein coding region of the Hprt gene. This additional gene, mor, not only serves to disrupt the target gene, rendering it inactive, but provides a marker for the selection of cells transformed by the targeting vector. Because most transformation protocols are quite inefficient, the vast majority of cells exposed to the targeting vector will not incorporate it. By tagging the vector with a marker gene, only those cells stably incorporating the vector will survive the screen for positive targeting events. A typical targeting experiment at Hprt proceeds as follows. The vector is introduced, generally by electroporation (Neumann et al., 1982; Chu et al., 1987), into a population of ES cells; the cells are divided into three aliquots grown under three growth conditions: 1) nonselectively, to assess the number of viable cells; 2 ) in the presence of G418, to assess the number of cells stably transformed with the vector; and 3 ) in media containing G418 and 6-TG, to select for those cells containing an Hprt gene disrupted by the neo' gene. This latter event can be confirmed at the gene level, by performing Southern transfer analysis on DNA extracted from G418', 6-Tff, cells. Typically, in a targeting experiment at the Hprt locus, there is one TARGETED MUTAGENESIS IN MURINE GENES ne0 A -4-Hx x I I I I I I 1 1 1 2 3 4 5 6 I I I I I I 1 2 3 4 5 6 I I I I 7/a 9 6 neo II 9 1 kb Figure 1. Targeted mutagenesis by sequence replacement of the Hprt locus. (A) Pairing of a targeting vector (top line) with the endogenous Hprt locus (bottom line). The vector contains DNA sequences from the murine Hprt locus in which the 7th and 8th exons have been replaced with the neor gene. Following recombination at positions indicated by X, endogenous sequences will be replaced by vector sequences. (B) The hprt- locus, resulting from homologous recombination as depicted in ( A ) . Thin lines represent introns and flanking sequences; solid boxes represent Hprt exons from 1 to 9; stippled boxes represent the mor gene. G41ar colony for every 100 cells surviving transformation (Thomas and Capecchi, 1987; Deng and Capecchi, 1992). Of those G41W colonies, 1 in 1,000 is also resistant to 6-TG. In all cases, those cells resistant to both drugs contain the predesigned mutation of the Hprt gene. Under optimum conditions, if one begins with lo5 cells, one can obtain one cell with the desired mutation. Because the frequency of homologous recombination depends on the enzymatic apparatus within the target cell, there are a limited number of parameters that can be vaned experimentally. Those parameters most easily varied involve manipulation of the targeting vector. Of the many variables in vector structure and topology that have been tested, only three seem to be of any consequence vis-&vis targeting frequency. First, the vector must be linear. The recombination machinery will apparently not recognize closed circular DNA (Thomas et al., 1986). Second, the DNA in the vector must be highly homologous to the target DNA. Mismatches of a few percent will severely decrease recombination, such that DNA from different mouse strains is not always recombinogenic (Deng and Capecchi, 1992; TeRiele et al., 1992). Third, the frequency of targeted recombination increases logarithmically with linear increases in the length of shared homology between vector and target. A twofold increase in homology can result in a 10- to 20-fold increase in targeting frequency (Thomas and 3 Capecchi, 1987; Deng and Capecchi, 1992). Unfortunately, this increase reaches saturation at a vector length of 12 to 15 kb of homology. Identification and clonal isolation of cells carrying targeted mutations at the Hprt locus present no severe problems to the investigator. However, this same luxury is not available to those interested in mutating the vast majority of other genes. The mutant phenotype will most likely be unknown (hence the rationale for performing the experiment), and may not be manifest in cultured cells. Because of this, selection protocols based on phenotype are difficult, if not impossible, to design. The most straightforward method for dealing with this obstacle is to screen for the targeted mutation by examining the DNA of all cells transformed with the targeting vector. This may, however, entail the analysis of several hundred cell lines, before the homologous recombinant can be identified among the random integrant siblings. An alternative is to reduce the number of nonhomologous recombinants that must be analyzed. This can be done, for example, by restricting the expressibility of the positive selectable marker. By eliminating the promoter sequences and(or) the 3' processing elements from the marker gene, the selectable marker will only be active when inserted into another active gene (Jasin and Berg, 1988). If the target gene is expressed in ES cells, it meets that requirement, and under such conditions, the ratio of uiable nonhomologoushomologous recombinants may be reduced by over 50-fold. A more versatile strategy, independent of the expression of the target locus, has also been devised to select against non-homologous recombinants. This strategy, termed positive-negative selection, relies on a difference in the exchange reactions mediating nonhomologous and homologous recombination (Mansour et al., 1988). The former generally occurs through the ends of the targeting vector, such that the entire vector is inserted into the genome. If a linear targeting vector contains on its ends DNA that is nonhomologous with the target, that DNA will be incorporated. Homologous exchange, however, only occurs over regions of homology, such that endterminal heterologous sequences will not be integrated. Thus, to decrease the viability of nonhomologous recombinants, a negative selectable marker can be included at the e n d ( s >of the targeting vector. Should the vector integrate by homology at the target, the negative marker will be eliminated and the cell remain viable. If the vector integrates at some random position via its ends, the marker is retained and the cell can be selected against. The most commonly used negative selectable marker is the thymidine kinase gene from the Herpes Simplex Virus. The enzyme encoded by this gene can convert a number of nucleotide analogues to products that are toxic to most mammalian cells, including mouse ES cells. Use of the positive-negative selection protocol 4 THOMAS neo 1 2 3 4 1 kb Figure 2. Mutant wnt-1- locus generated by gene targeting. The neoTgene has been inserted by homologous recombination into the 2nd exon of the Wnt-1 gene. Thin lines represent introns, solid boxes represent exons, stippled box represents the neo' gene. can enhance the ratio of homologoudnon-homologous recombinants by up t o 1,000-fold. Use of the positive-negative selection protocol has made it feasible to target mutations to virtually any gene in the mouse. Genes whose function is merely a matter of speculation can now be altered and the consequences of this alteration examined at the organismal level. This technology is particularly amenable to the study of genes presumed to be involved in development. Even if the gene is essential to the viability of the embryo, recessive alleles can be carried in heterozygous form and homozygosity created by a simple mating. The progeny from the intercross of heterozygous parents can then be examined for the mutant phenotype at all stages of embryogenesis. Targeted Mutagenesis of the Wnt-1 Gene The list of potential mammalian developmental genes is vast. It includes homologues of genes required for development of other organisms, genes encoding proteins known to act in tissue-specific fashion, genes whose mRNA product is developmentally regulated, and proto-oncogenes whose mis-expression or mutation affects growth and(or) differentiation. An example of one such gene, the indictment of which preceded by several years its conviction, is the Wnt-1 (Int-1) gene (reviewed in Nusse and Varmus, 1992). Identified first as an oncogene (Nusse and Varmus, 19821, which when ectopically induced by proviral insertion resulted in murine mammary carcinogenesis, the Wnt1 gene was later shown t o possess all the hallmarks of a candidate development gene. It is a member of a large gene family, consisting of 10 characterized relatives expressed in mice (Gavin et al., 1990; Nusse et al., 1991; Roelink and Nusse, 1991) and numerous homologues in species ranging from C. elegans to H. sapiens. The Drosophila Wnt-1 homologue is the wingEess (wg) gene, a well-Characterized member of the segment polarity class of genes and essential for normal development of the fruit fly (Morata and Lawrence, 1977; Rijsewijk et al., 1987). Furthermore, in mice, expression of the Wnt-1 is restricted develop- mentally to testes of adults and to dorsal midline cells in the developing neural tube (Jakobovits et al., 1986; Shackelford and Varmus, 1987; Wilkinson et al., 1987). Based on this restricted pattern of expression, it had been postulated that the Wnt-1 gene could be an essential component in nervous system development. This prediction has now been verified following the generation and analysis of mice carrying mutations in the Wnt-1 gene (McMahon and Bradley, 1990; Thomas and Capecchi, 1990). A mutation in the Wnt-I gene was created by use of the positive-negative selection strategy to introduce the 1-kb n e d gene into the second protein-coding exon of one Wnt-I allele in a mouse ES cell line. The site of this insertion mutation is between codon 54 and 55 of the 370-codon open-reading frame of the Wnt-I gene and is predicted t o cause a complete loss of function of the gene (see Figure 2). Embryo-derived stem cells heterozygous for the mutation were injected into wildtype mouse blastocysts that were then allowed to develop to term. One of the resulting chimeras was able to pass the mutation through the germ line and was used as a founder to establish a colony of mice heterozygous for the Wnt-I mutation. The phenotype of mice lacking a functional Wnt-1 gene is then ascertained following the mating of Wnt-1+/writ-1animals. The genotypes of progeny from such matings can be determined by DNA analysis of samples taken from both embryonic and adult tissue and correlated with the histological profile of the same individuals. One of the first questions asked following such a mating is whether the Wnt-I gene is required for viability of the embryo. In the analysis shown in Table 1, the number of embryos and newborn pups of each of the three possible genotypes is recorded as a function of days in utero. k o m a total of 30 litters, containing 223 progeny, the wild-type ( +/+)/heterozygote ( +/-I/ mutant ( -/-I ratio is remarkably close to the predicted Mendelian frequency of 1/2/1, suggesting nearly universal survival of embryos. It should be noted that two of the mutant fetuses examined at day 17.5 in Table 1. Genotype of embryos from heterozygous matings Days in utero No. of embryos of each genotype (no. of litters) 9.5 (2) 10.5 (2) 11.5 (2) 12.5 (2) 13.5 (3) 14.5 (1) 17.5 ( 5 ) Newborn ( 13) Total (30) ~ ~ aIncludes two dead. 2 6 3 1 5 3 13 23 56 6 12 11 11 12 3 18 43 116 6 0 4 5 5 2 ga 20 51 TARGETED MUTAGENESIS IN MURINE GENES Table 2. Viability of offspring from heterozygous matings Unanswered Questions No. of animals of each genotype ~~ Day of death 0-.5 +I+ .5-1.5 3 1 1.5-2.5 > 30 17 2 5 ~ +/- -I- 4 5 0 34 14 4 1 1 utero were dead, although no other obviously dead embryos were detected. In spite of the fact the homozygous mutants are generally viable throughout embryogenesis, they are frequently unable to survive to weaning age. As seen in Table 2, of 20 newborn animals genotyped as wnt-1-/wnt-l-, the vast majority died immediately following birth, and only one individual survived to adulthood (after additional matings, another wnt-I- adult has survived; see Thomas et al., 1991). Although this indicates some variability in expressivity of the wnt-l- mutation, it also identifies the Wnt-1 gene as an important component of normal mouse development. The one wnt-1-lwnt-1- offspring that sullrived to adulthood proved quite informative with respect to determining the site of action of the Wnt-1 gene (Thomas and Capecchi, 1990). The mutant animal showed a number of behavioral abnormalities, including ataxia, hypertonia, and circling movements, all of which are indicative of cerebellar abnormalities. Upon examination of mutant animals from embryonic d 9.5 t o adulthood, severe malformation of pre-cerebellar and cerebellar tissue was evident. As an example of this phenotype, sagittal sections from heterozygous and homozygous mutant individuals from embryonic d 11 and postnatal d 30 are shown in Figure 3. At the embryonic stage ( a and b ) the mutant shows severe underdevelopment of the metencephalon, the region a t the midbrain-hindbrain junction destined to become cerebellum, compared to its sibling. By adulthood ( c and d), this underdevelopment has been translated into a complete lack of the anterior cerebellar folia as well as midbrain hydroencephaly. Although all wnt-1-lwnt-1- animals showed malformations of the cerebellum, the mutation is highly variable in expressivity. Mutant individuals examined at birth, for example, fall roughly into two classes of equal number (Thomas et al., 1991). One-half of the animals have no identifiable cerebellar tissue and little of the characteristic midbrain nuclei, the superior and inferior colliculi. The other class have clearly visible midbrain nuclei (although their position is displaced) as well as some cerebellar tissue. One conclusion from this analysis is that the Wnt-I gene is minimally essential for the normal development of the anterior cerebellar compartment. This analysis identifies the stage of development that requires the Wnt-1 gene but leads to a number of interesting questions regarding the function of this gene. These same questions arise frequently when one analyzes other developmental mutants and thus deserve some consideration. The first is, What is the biochemical function of Wnt-1 gene product? Because of the stage of the first identifiable phenotype, embryonic d 9.5, a time of rapid proliferation of neuronal cells and their precursors, it is tempting to speculate that the wnt-I protein is required for the expansion of cells destined to populate the anterior cerebellum. The Wnt-I gene product has been shown in culture (and suggested by analogy to its Drosophila homologue, wg) to be a secreted protein (Heuvel et al., 1989; Papkoff and Schryvre, 1990; Nusse and Varmus, 1992), and is thus a candidate for a regionspecific mitogen. Such a designation awaits further investigation. A second question arising from this analysis is, Why is the wnt-l- phenotype restricted t o the cerebellar-midbrain region of the developing nervous system when the gene is expressed over a much greater portion of the dorsal neural tube (Wilkinson et al., 1987)? A number of answers include: 1) it is required elsewhere, but our detection techniques are too insensitive; 2 ) it is required elsewhere, but in its absence, other genes can compensate; the fact that expression of other members of the Wnt-gene family do overlap that of the Wnt-I gene makes them likely candidates for such complementing activity (Gavin et al., 1990); 3) expression must not necessarily correlate with gene function; assuming the Wnt-1 protein interacts with other proteins, functional activity depends on simultaneous expression of all interactive components. It should be emphasized that this lack of 100% correlation between gene expression and mutant phenotype is not restricted to wnt-1 mutants, but extends to a number of other developmental genes as well (Zijlstra et al., 1989; Koller et al., 1990; Soriano et al., 1991; Donehower et al., 1992). The final question posed by this analysis is that of expressivity. In the case of wnt-I- mice, a variability in expressivity is manifest in two forms: 1) viability, ranging from death in utero t o survival to adulthood, and 2 ) quantity of brain tissue, ranging from loss of the anterior cerebellum to loss of the entire cerebellum and some adjacent midbrain nuclei. In general there are three explanations for variability in expressivity: leakiness of the mutation, variable genetic background, and stochastic fluctuations in the activity of other genes. Partial activity of a mutant gene is always a possibility, unless of course the mutation is a complete deletion. In the case of the wnt-l- mutation, there are two arguments to suggest that the mutation is a null, loss-of-function allele. First, the position of the insertion of the mor gene should prohibit translation of the 6 THOMAS a +I d C --- *--* -1- Figure 3. Sagittal sections of wnt-l- mice (a and b). At 11.5 d embryos were fixed in Bouin’s reagent, embedded in paraffin, sectioned (10 p m ) ,and stained with hematoxylin and eosin (c and d). Brains were prepared as described previously (Thomas and Capecchi, 1990). Mid-sagittal sections (10 pm) stained with hematoxylin and eosin are shown. Genotypes were determined by analysis of DNA from either yolk sacs [a and b) or tails (c and d). met metencephalon; cf = cranial flexure; cb cerebellum; arrowhead in a and b indicates metencephalic region and in d the primary fissure. +theterozygous for wnt-1-; 7homozygous for wnt-1-. - - carboxy-terminal 80% of the Wnt-1 protein. Based on sequence analysis and comparisons with other Wnt1-gene family members, the untranslated portion of the protein is predicted t o contain a significant number of h c t i o n a l domains. The second evidence against the partial activity explanation is that a second, spontaneously occurring, mutant allele of wnt1 has recently been identified and shown also to vary in expressivity (Thomas et al., 1991). This allele, called swaying ( s w ) (Lane, 1967), contains a single base pair deletion that is predicted to cause premature termination of translation that would eliminate 50% of the Wnt-2 protein. Not only is the phenotype of sw homozygotes identical in all respects to that of the targeted mutant, but the compound heterozygote, containing both mutant alleles, exhibits the same variability in expressivity as well. The phenotypes caused by mutant alleles may be influenced by the genetic background of the animals in - - which they are expressed (Ramirez-Solis et al., 1993). The generation of targeted mutants often involves the outcrossing of two strains of mice, and the wnt-lallele has been maintained on a non-inbred background. To ascertain whether the extremes in phenotype could be explained by modifier loci present in one or more of the founder lines, the wnt-l- mice were crossed several times with different inbred and outcrossed lines of mice. No evidence of specific effector loci could be found (Thomas and Capecchi, unpublished data). Such variable expressivity of a mutant gene under a constant genetic background has been seen in several other instances as well. It seems likely, then, that the variability in expressivity seen in this and other cases (Chisaka et al., 1992;Mansour et al., 1993; Ramirez-Solis et al., 1993) may be the result of random variations in expression of genes involved in growth and development of tissues in the affected region. In the case of TARGETED MUTAGENESIS IN MURINE GENES the wnt-1- phenotype, for example, expansion of cells required for the posterior cerebellum and midbrain, the t w o structures variably affected, may involve two redundant pathways. In the absence of one of these, that containing Wnt-I, the other becomes the sole avenue for development of those structures. If activity of this other pathway is at or near some threshold requirement, it is likely that on occasion sufficient activity may not be provided. It is possible that such alternative pathways may be uncovered by analysis of mutations with phenotypes similar to those caused by the absence of the Wnt-I gene. The generation and analysis of mice deficient in the Wnt-I gene have demonstrated the potential of gene targeting in embryonic stem cells as a mechanism to study mammalian development. 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