Overview of Targeting by Homologous UNIT 4.29 Recombination

The analysis of mutant organisms and cell tion of the mutation present in the targeting lines has been important in determining the construct into the target gene. Once identified, function of specific proteins. Until recently, mutant ES cell clones can be microinjected into mutants were produced by mutagenesis fol- a normal blastocyst in order to produce a lowed by selection for a particular phenotypic chimeric . Because many ES cell lines change. Recent technological advances in gene retain the ability to differentiate into every cell targeting by in type present in the mouse, the can have mammalian systems enable the production of tissues, including the germ line, with contribu- mutants in any desired gene (Mansour, 1990; tion from both the normal blastocyst and the Robertson, 1991; Zimmer, 1992). This technol- mutant ES cells. Breeding germ-line chimeras ogy can be used to produce mutant mouse yields animals that are heterozygous for the strains and mutant cell lines. Because most mutation introduced into the ES cell, and that mammalian cells are diploid, they contain two can be interbred to produce homozygous mu- copies, or alleles, of each gene encoded on an tant mice. autosomal (nonsex) chromosome. In most Homologous recombination can also be cases, both alleles must be inactivated to pro- used to produce homozygous mutant cell lines. duce a discernible phenotypic change in a mu- Previously, inactivation of both alleles of a gene tant. The conversion from heterozygosity to required two rounds of homologous recombi- homozygosity is accomplished by breeding in nation and selection (te Riele et al., 1990; Cruz the case of mouse strains and by direct selection et al., 1991; Mortensen et al., 1991). Now, in cell lines. however, inactivation of both alleles of many recombinases such as Cre requires only a single round of homolo- and its recognition sequence, loxP, have also gous recombination using a single targeting allowed spatial control of knockouts. Another construct (Mortensen et al., 1992). The ho- recombinase system, the Flp/FRT system, mozygous mutant cells can then be analyzed can also be used (Fiering et al., 1993, 1999). for phenotypic changes to determine the func- The control can function along actual spatial tion of the gene. coordinates when a viral gene transfer system is used, or in a cell type- or -specific ANATOMY OF TARGETING fashion when restricted promoters are em- CONSTRUCTS ployed. Adding temporal regulation of Cre, Two basic configurations of constructs are such as that achievable with the tetracycline- used for homologous recombination: insertion regulatable expression system (see CPMB UNIT constructs and replacement constructs (Fig. 16.21 and APPENDIX 1A in this manual), allows 4.29.1). Each can be used for different pur- temporal control as well. poses in specific situations, as discussed be- To produce a mutant mouse strain by ho- low. The insertion construct contains a region mologous recombination, two major elements of homology to the target gene cloned as a are needed. An embryonic stem (ES) cell line single continuous sequence, and is linearized capable of contributing to the germ line, and a by cleavage of a unique restriction site within targeting construct containing target-gene se- the region of homology. Homologous recom- quences with the desired mutation. Maintain- bination introduces the insertion construct se- ing ES cells in their undifferentiated state is a quences into the homologous site of the target major task during gene targeting (see CPMB UNIT gene, interrupting normal target-gene struc- 23.3 and APPENDIX 1A in this manual). This usually ture by adding sequences. As a result, the is accomplished by growing cells on a layer of normal gene can be regenerated from the mu- feeder cells (see CPMB UNIT 23.2 and APPENDIX 1A tated target gene by an intrachromosomal re- in this manual). The targeting construct is then combination event. transfected into cultured ES cells (see UNIT 4.30). The replacement construct is the second, ES cell lines are derived from the inner cell more commonly used construct. It contains two mass of a blastocyst-stage . Homolo- regions of homology to the target gene located gous recombination occurs in a small number on either side of a mutation (usually a positive Gene , of the transfected cells, resulting in introduc- selectable marker; see below). Homologous Expression, and Mutagenesis Contributed by Richard Mortensen 4.29.1 Current Protocols in Neuroscience (2002) 4.29.1-4.29.10 Copyright © 2002 by John Wiley & Sons, Inc. Supplement 21 insertion construct replacement construct neo neo ∗ ∗ ∗ ∗ 2 3 2 3

1 2 3 4 1 2 3 4

neo neo ∗ ∗ ∗ ∗ 1 2 3 2 3 4 1 2 3 4

Homologous sequences Nonhomologous sequences construct and target gene positive selectable marker target gene vector

Figure 4.29.1 Two configurations of constructs used for homologous recombination. Numbers indicate target-gene sequences in the . An asterisk indicates homologous target-gene sequences in the construct. Replacement constructs substitute their sequences (2*, neo, and 3*) for the endogenous target-gene sequences (2 and 3). Insertion constructs add their sequences (2*, neo, and 3*) to the endogenous target gene, resulting in tandem duplication and disruption of the normal gene structure.

recombination proceeds by a double cross-over Positive-Negative Selection event that replaces the target-gene sequences The most commonly used method for elimi- with the replacement-construct sequences. Be- nating cells in which the construct integrated cause no duplication of sequences occurs, the into the genome randomly, thus further enrich- normal gene cannot be regenerated. ing for homologous recombinants, is known as positive-negative selection. It is only applicable METHODS OF ENRICHMENT FOR to replacement constructs (Fig. 4.29.2; Man- HOMOLOGOUS RECOMBINANTS sour et al., 1988). In these constructs, a negative selectable marker (e.g., herpes simplex Positive Selection by Drug-Resistance , HSV-TK) is included outside Gene the region of homology to the target gene. In Nearly all constructs used for homologous the presence of the TK gene, the cells are recombination rely on the positive selection sensitive to acyclovir and its analogs (e.g., of a drug-resistance gene (e.g., neomycin or gancyclovir, GANC). The HSV-TK neo) that is also used to interrupt and mutate activates these drugs, resulting in their incor- the target gene. When either insertion or re- poration into growing DNA, causing chain ter- placement constructs are linearized, the drug- mination and cell death. During homologous resistance gene is flanked by two regions of recombination, sequences outside the regions homology to the target gene. Selection of the of homology to the target gene are lost due to cells using drugs (e.g., G418) eliminates the crossing over. In contrast, during random inte- great majority of transformants that have not gration all sequences in the construct tend to be stably incorporated the construct (see CPMB UNIT retained because recombination usually occurs 9.5 and APPENDIX 1A in this manual). However, in at the ends of the construct. The presence of the many of the surviving clones the construct has TK gene can be selected against by growing the incorporated into the genome not by homolo- cells in gancyclovir; the homologous recombi- gous recombination but rather through random nants will be G418-resistant and gancyclovir- Overview of Gene integration. Therefore, methods to enrich for resistant, whereas clones in which the construct Targeting by Homologous homologous recombinant clones have been de- integrated randomly will be G418-resistant and Recombination veloped. gancyclovir-sensitive. In some cases, TK is 4.29.2

Supplement 21 Current Protocols in Neuroscience homologous recombination random integration

neo TK neo TK ∗ ∗ ∗ ∗ 2 3 2 3

1 2 3 4

neo neo TK ∗ ∗ ∗ ∗ 1 2 3 4 2 3

cell phenotype: G418R G418R GANCR GANCS

Homologous sequences Nonhomologous sequences construct and target gene positive selectable marker target gene negative selectable marker vector promoter

Figure 4.29.2 Enrichment for homologous recombinants by positive-negative selection using the TK gene. Homologous recombination involving cross-overs on either side of the neo gene results in loss of the TK gene. Random integration tends to preserve the TK gene. The presence of TK can be selected against because any cell expressing the gene will be killed by gancyclovir (GANC). Although both homologous recombinants and clones in which the construct integrated randomly are G418-resistant, only homologous recombinants are gancyclovir-resistant. The construct is shown linearized so that the plasmid vector sequences remain attached to the TK gene. This configuration helps preserve the integrity of the TK gene. The superscript R denotes resistance and the superscript S denotes sensitivity. inactivated without homologous recombina- recombination occurs, a fusion protein is pro- tion; thus, the gancyclovir-resistant clones duced, driven by the endogenous target-gene must be screened to identify the true homolo- promoter. In contrast, when random integration gous recombinants. Other markers that are le- occurs, the selectable-marker protein is not thal to cells have also been used instead of TK usually produced. Therefore, homologous re- and gancyclovir (e.g., diphtheria toxin; Yagi et combinants are G418-resistant, whereas cells al., 1990). in which the construct integrated randomly are G418-sensitive. Constructs containing a pro- Endogenous Promoters moterless selectable marker can be constructed Constructs that rely on an endogenous pro- in either replacement or insertion structure and moter to express the positive selectable marker can result in dramatic enrichment for homolo- can also give enrichment of homologous re- gous recombinants. combinants (Fig. 4.29.3), but can only be used if the gene of interest is expressed in the cell TYPES OF MUTATIONS line. They contain the coding region of a select- able marker (e.g., neo) but lack a promoter for Gene Inactivation the marker. The coding sequence for the marker Homologous recombination has most often usually interrupts, and is in frame with, an been used to completely inactivate a gene (com- Gene Cloning, Expression, and of the target gene. Thus, when homologous monly termed “knockout”). Usually, an exon Mutagenesis 4.29.3

Current Protocols in Neuroscience Supplement 21 homologous recombination random integration

neo neo

exon 1 exon 2 exon 3

neo

mRNA no product

fusion protein

cell phenotype: G418R G418S

Homologous sequences Nonhomologous sequences construct and target gene positive selectable marker target gene promoter

Figure 4.29.3 Enrichment for homologous recombinants using a positive selectable marker (neo) lacking a promoter. Clones in which integration of the construct provides an endogenous promoter to drive neo expression will be G418-resistant. The construct is designed so that homologous recombination will provide a promoter leading to neo expression, whereas random integration will most likely not provide a promoter, thus precluding neo expression.

encoding an important region of the protein (or Mutations can be introduced that have mul- an exon 5′ to that region) is interrupted by a tiple purposes. Homologous recombination has positive selectable marker (e.g., neo), preventing been used to introduce a replacement construct the production of normal mRNA from the target containing the coding sequence of β-galactosi- gene and resulting in inactivation of the gene. dase in frame with the 5′ end of the target gene. A gene may also be inactivated by creating Downstream of the lacZ gene is a positive a deletion in part of a gene, or by deleting the selectable marker driven by a heterologous pro- entire gene. By using a construct with two moter (Fig. 4.29.4). This construct not only regions of homology to the target gene that are disrupts target-gene function but also expresses far apart in the genome, the sequences interven- a fusion protein with β-galactosidase activity, ing the two regions can be deleted. Up to 15 kb and thus can be used to monitor the activity of have been deleted in this way; thus, many genes the endogenous gene’s promoter in various tis- could be completely eliminated (Mombaerts et sues during development (Mansour et al., al., 1991). Gene inactivations may also be con- 1990). trolled using the Cre/loxP recombinase system either spatially, as in cell type- or tissue-specific Subtle Gene Mutations Overview of , or temporally, through control of the Homologous recombination can also be Targeting by activity or expression of the recombinase (see used to introduce subtle mutations in a gene. Homologous Cre/loxP System, below). One method is analogous to a method in yeast Recombination 4.29.4

Supplement 21 Current Protocols in Neuroscience lacZ neo

exon 1 exon 2 exon 3

lacZ neo

mRNA

fusion protein

Homologous sequences Nonhomologous sequences construct and target gene positive selectable marker target gene lacZ gene vector promoter

Figure 4.29.4 LacZ reporter construct for gene targeting. This construct has two purposes: first, to disrupt the target gene and, second, to express the lacZ gene as a marker to monitor activity of the endogenous target gene’s promoter. called transplacement or allele replacement that eliminates the target-gene duplications and (CPMB UNIT 13.10). It is called “hit and run” (see the selectable markers but leaves the mutant CPMB UNIT 13.10 and APPENDIX 1A in this manual) target-gene sequences substituting for the nor- because duplications are introduced into the mal target-gene sequences. Surviving clones are target gene and then removed. An insertion screened for the correct intrachromosomal rear- construct containing both positive and negative rangements, leaving the desired mutation. A selectable markers (e.g., neo and TK) is used second method of introducing subtle mutations to introduce a duplication that contains a subtle into a gene is to insert the mutation by homolo- mutation, such as a , into the gous recombination and then use the Cre/loxP target gene sequence (Fig. 4.29.5). After selec- system to remove the selectable marker. tion for integration of the construct using the positive selectable marker (e.g., G418), ho- CRE/loxP SYSTEM mologous recombinants are identified by The Cre/loxP system is derived from the screening. A homologous recombinant clone is bacteriophage P1. The recombinase Cre acts on cultured and then the presence of the negative the DNA site loxP. If there are two loxP sites selectable marker is selected against (e.g., se- in the same orientation near each other, Cre can lection against TK using gancyclovir). This act to loop out the sequence between the two Gene Cloning, selects for an intrachromosomal recombination sites, leaving a single loxP site in the original Expression, and Mutagenesis 4.29.5

Current Protocols in Neuroscience Supplement 22 TK neo † ∗ ∗ 2 3

1 2 3 4

select G418

neo ∗ ∗ 1 2 3 2 3 4

† ∗ 1 2 3

∗ 2 3 4

select GANC

† ∗ 1 2 3 4

Homologous sequences Nonhomologous sequences construct and target gene positive selectable marker target gene negative selectable marker vector promoter

Figure 4.29.5 Allele replacement “hit and run.” Cells are cultured in G418 to select for the integration of neo, then homologous recombinants are identified by screening and are cultured in gancyclovir (GANC) to select against the presence of the TK gene. This strategy may yield a reconstituted gene containing the subtle mutation present in the construct (indicated by the dark bar and †). Because intrachromosomal recombination may result in the loss of the subtle mutation, its presence must be verified (e.g., by a change in restriction site).

DNA and a second loxP in a circular piece of Removing the Positive Selectable DNA containing the intervening sequence. Marker Therefore, a properly designed targeting con- Although many gene inactivation ap- struct containing loxP sites can be used for proaches involving homologous recombina- introducing subtle mutations or for a tempo- tion still use constructs that leave the positive rally or spatially controlled knockout (for a selectable marker in the genomic DNA, it has review of the control of , see Sauer, become increasingly clear that this can cause a 1993). number of unanticipated effects. For example, Overview of Gene Other recombinase systems, such as the the presence of the neo gene, often with its own Targeting by Flp/FRT system, can be similarly useful (Fier- promoter, can alter the expression of neighbor- Homologous ing et al., 1995; Vooijs et al., 1998). ing loci (Olson et al., 1996; Pham et al., 1996). Recombination 4.29.6

Supplement 22 Current Protocols in Neuroscience ∗

Cre expression

∗

homologous recombination ∗

Homologous seqeunces Nonhomologous sequences construct and target gene positive selectable marker genomic sequences lox sequence vector sequences promoter sequence mutation ∗

Figure 4.29.6 Using the Cre/loxP system to introduce subtle mutations. The subtle mutation is introduced along with the selectable marker in the targeting vector. The selectable marker is then removed by transient expression of Cre, which leaves only the small loxP site in the genome in a silent location.

This can be a particular problem in gene clus- tion in vivo, where the expression of Cre is ters where neighboring genes are in the same derived from sequences integrated into the family, since the genes affected may have simi- genome and therefore will show longer-lasting lar or identical functions. As a result, slight expression in nearly all cases. differences in targeting constructs have led to marked differences in phenotype. Introduction of Subtle Mutations If the targeting construct includes loxP sites Using Cre/loxP flanking the neo gene, then neo can be removed The strategy described in the previous sec- after targeting by transient expression of the tion involves introducing subtle mutations by (as discussed in Fig. 4.29.3). first duplicating sequences and then screening This will leave the small loxP site in the for intrachromosomal recombination that re- genomic DNA, but the construct can be engi- moves the redundant sequences and leaves the neered so that this is in an innocuous location, mutation. A limitation to this approach is that such as an . Although theoretically even the second homologous recombination event a loxP site could cause alterations in the expres- occurs only infrequently. A more efficient sion of neighboring genes, no such cases have method is to use a replacement construct con- yet been reported. The efficiency of Cre recom- taining the subtle mutation and then remove the bination from transient expression reported in positive selectable marker, which is flanked by the literature varies widely, from ∼2% to ∼15% loxP sites, using the Cre recombinase system (Sauer and Henderson, 1989; Abuin and (Fig. 4.29.6). This is identical in effect to re- Gene Cloning, Bradley, 1996). This rate should be distin- moving the neo locus after gene inactivation, Expression, and guished from the efficiency of Cre recombina- except that instead of an inactive gene, the Mutagenesis 4.29.7

Current Protocols in Neuroscience Supplement 21 Cre expression or or in culture

or or

homologous recombination

Cre expression in vivo

Homologous sequences Nonhomologous sequences construct and target gene positive selectable marker genomic sequences lox sequence vector sequences promoter sequence

Figure 4.29.7 Conditional gene targeting using the Cre/loxP system. The targeting vector contains three loxP sites that flank the regions of the gene to be removed and the positive selectable marker neo. After homologous recombination is obtained, the selectable marker is excised from the gene by transient expression of Cre. The correct recombination is identified by screening by Southern analysis or PCR. The mutant ES cells are then used to produce a transgenic animal, which is finally bred to an animal line expressing Cre under either temporal or spatial control. Cre can also be ubiquitously expressed to obtain a knockout in all tissues, including the germ line.

replaced sequences contain a subtly mutated using a cell type-restricted promoter. A second version. transgenic animal line is then created by ho- mologous recombination that contains loxP Spatial Control of Knockout sites flanking a portion of the gene that is Spatially controlled targeted gene inactiva- critical for activity, typically important tions can be performed in two ways. The most (Fig. 4.29.7). Initially there are three loxP sites common makes use of cell type-specific pro- flanking this important gene region and the moters (sometimes called tissue-specific pro- selectable marker. After homologous recombi- moters, even though tissues are actually made nation has been verified, Cre is transiently ex- Overview of Gene of a number of different cell types). This ap- pressed, and loops out regions of DNA between Targeting by Homologous proach begins with the creation of a transgenic pairs of loxP sites. The resultant colonies are Recombination animal that expresses Cre in only some cells screened for the desired recombination (loss of 4.29.8

Supplement 21 Current Protocols in Neuroscience the selectable marker but retention of all re- 1996) or other inducible system (such as an gions of the gene). Depending on the frequency interferon-inducible promoter; Kuhn et al., of recombination at the site, it may be useful to 1995) to express Cre at the proper time. This use a construct that contains a negative select- would, however, require the construction of able marker (such as cytosine deaminase in the animals containing three transgenes. Another example shown in Fig. 4.29.2) between the approach that has been used is the creation of loxP sites along with the positive selectable a fusion protein with either a modified estrogen marker. In this way cells that have lost the receptor (Feil et al., 1996, 1997; Zhang et al., markers can be selected. 1996; Brocard et al., 1997) or a modified glu- The targeted line will have normal expres- cocorticoid receptor (Brocard et al., 1998). sion of the targeted gene, since its only modi- These fusion proteins are inactive for recombi- fication is the presence of loxP sites in innocu- nation until the appropriate ligand is added, ous sites (e.g., ). When the two lines are allowing temporal control in an animal with bred together, the Cre recombinase will loop only transgenes. The Flp/FRT recombinase out the DNA—inactivating the gene—only in system can be used in an analogous way. Com- those cells where it is expressed. In this way, bination of the two systems can allow the pro- tissue-specific knockouts of a number of genes duction of complex schemes for gene mutation. have been generated (Gu et al., 1994; Agah et al., 1997). The method also has the advantage LITERATURE CITED that, once a transgenic line is generated with Abuin, A. and Bradley, A. 1996. Recycling selectable the desired restricted expression of Cre, the markers in mouse embryonic stem cells. Mol. approach can be applied to a number of targeted Cell. Biol. 16:1851-1856. lines. In addition, it is not necessary to make Agah, R., Frenkel, P.A., French, B.A., Michael, L.H., separate constructs for a restricted and a com- Overbeek, P.A., and Schneider, M.D. 1997. Gene recombination in postmitotic cells. Targeted ex- plete knockout, since Cre-expressing lines have pression of Cre recombinase provokes cardiac-re- been made that will produce rearrangement in stricted, site-specific rearrangement in adult ven- all tissues when bred to the targeted line tricular muscle in vivo. J. Clin. Invest. 100:169- (Schwenk et al., 1995). 179. Another way of spatially controlling knock- Brocard, J., Warot, X., Wendling, O., Messaddeq, N., out is to use an expression system for Cre that Vonesch, J.L., Chambon, P., and Metzger, D. can be applied to absolute location. In some 1997. Spatio-temporally controlled site-specific somatic mutagenesis in the mouse. Proc. Natl. cases, no restricted expression pattern is known Acad. Sci. U.S.A. 94:14559-14563. for a gene that matches the desired spatial Brocard, J., Feil, R., Chambon, P., and Metzger, D. alteration; in others, the site may be particularly 1998. A chimeric Cre recombinase inducible by amenable to viral manipulation (as with an synthetic, but not by natural ligands of the gluco- epithelial or endothelial surface) or accessible corticoid receptor. Nucl. Acids Res. 26:4086- by direct injection (such as sterotactic injection 4090. of the central nervous system). By using a viral Cruz, A., Coburn, C.M., and Beverley, S.M. 1991. vector to express the Cre protein, it is possible Double targeted gene replacement for creating to obtain knockouts that are spatially limited null mutants. Proc. Natl. Acad. Sci. U.S.A. 88:7170-7174. by the viral infection. This strategy has been applied to a number of tissues including the Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and Chambon, P. 1996. Ligand-ac- brain, liver, colon, and heart (Rohlmann et al., tivated site-specific recombination in mice. Proc. 1996; Wang et al., 1996; Agah et al., 1997; Natl. Acad. Sci. U.S.A. 93:10887-10890. Shibata et al., 1997; van der Neut, 1997). Feil, R., Wagner, J., Metzger, D., and Chambon, P. 1997. Regulation of Cre recombinase activity by Temporal Control of Knockout mutated estrogen receptor ligand-binding do- In many cases the phenotype of interest is mains. Biochem. Biophys. Res. Commun. in the adult animal but, because the gene is 237:752-757. necessary for development, no adult animals Fiering, S., Kim, C.G., Epner, E.M., and Groudine, are obtained. Delaying the expression of Cre M. 1993. An “in-out” strategy using gene target- ing and FLP recombinase for the functional dis- activity until the animal is an adult would allow section of complex DNA regulatory elements: normal development, and then the knockout Analysis of the β-globin locus control region. could be created in the adult (Rajewsky et al., Proc. Natl. Acad. Sci. U.S.A. 90:8469-8473. 1996). This can be accomplished by using a Fiering, S., Epner, E., Robinson, K., Zhuang, Y., Gene Cloning, conditional expression system (e.g., the tet-on, Telling, A., Hu, M., Martin, D.I., Enver, T., Ley, Expression, and tet-off, or ecdysone systems; see St-Onge et al., T.J., and Groudine, M. 1995. Targeted deletion Mutagenesis 4.29.9

Current Protocols in Neuroscience Supplement 21 of 5′HS2 of the murine β-globin LCR reveals gene inactivation by viral transfer of Cre recom- that it is not essential for proper regulation of the binase. Nat. Biotechnol. 14:1562-1565. β -globin locus. Genes Dev. 9:2203-2213. Sauer, B. 1993. Manipulation of transgenes by site- Fiering S., Bender, M.A., and Groudine, M. 1999. specific recombination: Use of Cre recombinase. Analysis of mammalian cis-regulatory DNA ele- Methods Enzymol. 225:890-900. ments by homolgous recombination. Methods Sauer, B. and Henderson, N. 1989. Cre-stimulated Enzymol. 306:42-66. recombination at loxP-containing DNA se- Gu, H., Marth, J.D., Orban, P.C., Mossmann, H., and quences placed into the mammalian genome. Rajewsky, K. 1994. Deletion of a DNA polym- Nucl. Acids Res. 17:147-161. β erase gene segment in T cells using cell type– Schwenk, F., Baron, U., and Rajewsky, K. 1995. A specific gene targeting. 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Olson, E.N., Arnold, H.H., Rigby, P.W., and Wold, 1990. Homologous recombination at c-fyn locus B.J. 1996. Know your neighbors: Three pheno- of mouse embryonic stem cells with use of diph- types in null mutants of the myogenic bHLH gene theria toxin A-fragment gene in negative selec- MRF4. Cell 85:1-4. tion. Proc. Natl. Acad. Sci. U.S.A. 87:9918-9922. Pham, C.T., MacIvor, D.M., Hug, B.A., Heusel, J.W., Zhang, Y., Riesterer, C., Ayrall, A.M., Sablitzky, F., and Ley, T.J. 1996. Long-range disruption of gene Littlewood, T.D., and Reth, M. 1996. Inducible expression by a selectable marker cassette. Proc. site-directed recombination in mouse embryonic Natl. Acad. Sci. U.S.A. 93:13090-13095. stem cells. Nucl. Acids Res. 24:543-548. Rajewsky, K., Gu, H., Kuhn, R., Betz, U.A., Muller, Zimmer, A. 1992. Manipulating the genome by ho- W., Roes, J., and Schwenk, F. 1996. Conditional mologous recombination in embryonic stem gene targeting. J. Clin. Invest. 98:600-603. cells. Annu. Rev. Neurosci. 15:115-137. Robertson, E.J. 1991. Using embryonic stem cells to introduce mutations into the mouse germ line. Contributed by Richard Mortensen Biol. Reprod. 44:238-245. University of Michigan Rohlmann, A., Gotthardt, M., Willnow, T.E., Ham- Medical School mer, R.E., and Herz, J. 1996. Sustained somatic Ann Arbor, Michigan Overview of Gene Targeting by Homologous Recombination 4.29.10

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