Recent advances in gene mutagenesis by site-directed recombination. J D Marth J Clin Invest. 1996;97(9):1999-2002. https://doi.org/10.1172/JCI118634. Perspective Find the latest version: https://jci.me/118634/pdf Perspectives Series: Molecular Medicine in Genetically Engineered Animals Recent Advances in Gene Mutagenesis by Site-directed Recombination Jamey D. Marth Howard Hughes Medical Institute, Department of Medicine and Division of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093 Transgenic experimentation yields insights that could not be lack of cell or tissue development may result from ES cell perceived otherwise among populations of mammalian organ- clonal variation, use of multiple ES clones and complementa- isms (reviewed in references 1 and 2). However, information tion by gene transfer allow for controlled studies. Some con- gained may, on occasion, be somewhat limited should germ- siderations remain the inability to direct genetic variation to line gene dysfunction be deleterious in embryonic develop- multiple, experimentally defined cell lineages and the poten- ment, thereby precluding analyses in various somatic compart- tially unique nature of each chimeric mouse generated. ments of the adult organism. The production of chimeric mice Since inception, gene transfer experimentation has been bearing genetic mutations specifically in various somatic cells generally limited to irreversible modifications of chromosomal allows the study of gene function in physiologic contexts that DNA. Isolation of cells having undergone site-specific ex- would otherwise be unavailable or lethal. One elegant cell change or deletion of DNA sequence has only recently been type-restricted chimeric approach has been the use of the developed using novel screening strategies for often low fre- RAG 2-null embryos as recipients in gene-targeted embryonic quency events. To develop methods allowing high frequency stem (ES)1 cell–derived lymphoid development (3). While a site-specific chromosomal DNA modification, several labora- tories have been investigating the activities of DNA recombi- nase enzymes to provide for additional experimental control Address correspondence to Jamey D. Marth, Howard Hughes Medi- over gene transfer and resulting mutagenesis among cell popu- cal Institute, 9500 Gilman Drive, 0625, University of California San lations. Among the integrase family of recombinases, one of Diego, La Jolla, CA 92093. Received for publication 15 February 1996 and accepted in revised current relevance is termed Cre and exists as a 30-kD enzyme form 6 March 1996. encoded within the genome of bacteriophage P1 (4). Recombi- nation by Cre excises DNA residing between direct repeats of 1. Abbreviations used in this paper: CMV, cytomegalovirus; ES, em- a 34-bp DNA substrate termed loxP. Cre recombination does bryonic stem; tet, tetracycline. not require ATP or topoisomerase activity (5) and has been found to function in heterologous eukaryotic genomes, includ- “Molecular Medicine in Genetically Engineered Animals” Series Editor, Kenneth R. Chien January 1 Gene modification via “plug and socket” gene targeting................................. Jada Lewis, Baoli Yang, Biological insights through genomics: mouse to man ..................................... Pete Detloff, and Oliver Smithies January 15 Biological insights through genomics: mouse to man ..................................... Edward M. Rubin and Gregory S. Barsh February 1 In vitro differentiation of murine embryonic stem cells: new approaches to old problems .................................................................... Mitchell J. Weiss and Stuart H. Orkin February 15 Genes and physiology: molecular physiology in genetically engineered animals......................................................................................... Kenneth R. Chien March 1 Animal models of human disease for gene therapy........................................ James M. Wilson March 15 Targeted mutagenesis: analysis of phenotype without germ line transmission.................................................................................................... Andras Nagy and Janet Rossant April 1 Transgenesis in the rat and larger mammals.................................................. Linda J. Mullins and John J. Mullins April 15 The zebrafish: heritable diseases in transparent embryos ............................. Wolfgang Driever and Mark Fishman May 1 Recent advances in gene mutagenesis by site-directed recombination............................................................................. Jamey Marth J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/96/05/1999/04 $2.00 Volume 97, Number 9, May 1996, 1999–2002 Recent Advances in Gene Mutagenesis by Site-directed Recombination 1999 ing mammalian cell lines (6, 7). Another member of the inte- Beginning with a cloning vector bearing three loxP sites, grase family is termed Flp and is encoded within the 2 ␮M cir- two of which flank the selectable marker genes Neo and HSV- cle of yeast (8). Flp recombinase acts identically to Cre and TK, a targeting construct is generated that allows for the pro- recognizes a similar DNA sequence substrate termed frt. Both duction of systemic and conditional gene mutagenesis models use a transient high energy covalent peptide-DNA intermedi- after homologous recombination (Fig. 1). loxP-containing ate in DNA strand cleavage and ligation reactions. Flp has also isogenic gene targeting constructs thus designed and with sev- been found to function in the genomes of Drosophila melano- eral kilobases of genomic DNA have yielded homologous re- among G418-resistant 0.025 ف gaster and some mammalian cell lines after gene transfer (9, combination frequencies of 10), although for reasons thus far uncertain, Cre recombinase clones in experiments with nine different gene loci (unpub- appears to function at significantly higher efficacy in mamma- lished data). While inclusion of loxP sites may not affect the lian cells. frequency of homologous recombination, use of a loxP-spe- of %50 ف Transgenic studies of Cre recombinase activity previously cific probe in genomic Southern blotting reveals that reported that Cre could act in an efficient, heritable, tissue-, gene-targeted ES clones lack the loxP site most distal from the and site-specific manner to excise DNA specifically flanked by Neo and HSV-TK cassette apparently as a result of crossover direct repeats of loxP at distinct chromosomal locations (11, events specifically within the loxP-flanked genomic sequence 12). After recombination, excised DNA was degraded as it was (16, and unpublished data). ES cell clones that have under- not maintained extrachromosomally or found integrated else- gone homologous recombination and retain all three loxP sites -␮g of Cre expression vector in su 1 ف where in the genome. Recombination and deletion of DNA are electroporated with residing between two direct repeats of loxP resulted in the percoiled plasmid form. ES cell sub-clones are then isolated af- elimination and activation of gene function, in the latter case ter a 5–7 d selection in the presence of 0.5–2 ␮M gancyclovir by excision of a “stop” element that otherwise blocked expres- begun 48–72 h after electroporation. In experience, virtually sion of 3Ј sequences. The assessed efficacy of Cre recombina- all resulting gancyclovir-resistant clones isolated have under- tion was quite high, providing the expectation of using Cre- gone either a Type I or Type II recombination yielding ES mediated recombination effectively for conditional gene mu- cells required for producing the systemic and conditional mu- tagenesis techniques in embryonic stem cells and gene-tar- tations in vivo, respectively (Fig. 1), often with the majority ex- geted mice. That potential was initially achieved with the in- hibiting the Type I deletion. However, gancyclovir resistant corporation of functional recombinase target sites into the clones may also arise as a result of wild-type ES cell contami- endogenous mouse immunoglobulin locus using homologous nants and from those cells bearing deleterious HSV-TK muta- recombination in ES cells (13, 14). Subsequently, a novel tar- tions. Several ways to increase the frequency of Type II recom- geting strategy was devised in which three loxP sites are used bination events are possible: one can reduce the amount of Cre during homologous recombination to acquire ES cell clones vector used in the electroporation, use less efficient promoters ultimately lacking the selectable markers and yet bearing ei- to express Cre, and use Cre DNA sequence that lacks nuclear ther systemic or conditional gene mutations upon mouse gen- localization and eukaryotic translational consensus signals (see eration (15, and see below). These studies have developed a below). method enabling tissue-specific gene mutagenesis in vivo. Be- After the production of chimeric and heterozygous mice low, relevant experiences using this technique are described as from ES cell clones containing the Type II recombination, a well as expected advances in the near future. breeding strategy to achieve conditional gene mutation in vivo Figure 1. Systemic and conditional mutagenesis in embryonic stem cells as generated after homolo- gous recombination and transient Cre recombinase expression. The frequency of Type I deletions is ten times higher than that for Type II deletions (see text). 34-bp loxP sites are depicted as shaded