© 2000 Wiley-Liss, Inc. genesis 26:99–109 (2000) REVIEW ARTICLE Cre Recombinase: The Universal Reagent for Genome Tailoring Andras Nagy* Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada Received 8 November 1999; Accepted 9 November 1999 INTRODUCTION BASIC PROPERTIES OF CRE RECOMBINASE In September 1998 the workshop for “Conditional Ge- The Cre recombinase of the P1 bacteriophage belongs to nome Alterations” in Cold Spring Harbor had a simple the integrase family of site-specific recombinases. It is a but important take-home message: “Cre works” (Rossant 38 kD protein that catalyzes the recombination between and McMahon, 1999). After several years of careful in- two of its recognition sites, called loxP (Hamilton and vestigations, geneticists are now certain that the Cre Abremski, 1984). This is a 34 bp consensus sequence, recombinase of the P1 bacteriophage efficiently cata- consisting of a core spacer sequence of 8 bp and two 13 lyzes recombination between two of its consensus 34 bp palindromic flanking sequences (Fig. 1A). The asym- base pair DNA recognition sites (loxP sites) in any cel- metric core sequence defines an orientation to the loxP lular environment and on any kind of DNA. Obviously site. One of the great advantages of the Cre/loxP recom- mouse geneticists, including myself, are among those who bination system is that there is no need for additional are most excited about this finding. The reason is obvious: co-factors or sequence elements for efficient recombina- When we acquired the Cre/loxP recombinase system as a tion regardless of the cellular environment. Concerning tool, we reached the level of sophistication of “no more the molecular mechanism of recombination, a single practical limitation” in tailoring the mouse genome. recombinase molecule binds to each palindromic half of The well-established practice of transgenesis allows a loxP site, then the recombinase molecules form a the addition of functional DNA pieces into the mouse tetramer, thus bringing two loxP sites together (Voziy- genome. Furthermore, with the advent of mouse embry- anov et al., 1999). The recombination occurs within the onic stem (ES) cells and homologous recombination- spacer area of the loxP sites. The postrecombination based gene targeting, we are capable of removing any loxP sites are formed from the two complementary existing gene from the genome. By adding the Cre re- combinase system, we can excise or invert loxP-flanked halves of the prerecombination sites (Fig. 1A). DNA segments or create intermolecular recombination The first indication that the phage enzyme would between different DNA molecules. By combining all of work in eukaryotic cells emerged from in vitro cell these tools, we are capable of creating any desired mod- culture experiments in the late ’80s and early ’90s (Sauer ification of the mouse genome, from introducing specific and Henderson, 1988; 1989; 1990). These experiments point mutations to large site-specific chromosomal aber- were followed by the demonstration that the Cre recom- rations. Furthermore, using the recombinase activity as a binase worked in the mouse as well when expressed genetic activation or inactivation switch, conditional from a transgene (Lakso et al., 1992; Orban et al., 1992). An important issue is that a random occurrence of a transgenesis or conditional knock-outs became available. 18 The strategies are designed; the proof of principles are specific 34 bp sequence requires a 10 bp length of ϫ 9 given. Many of these strategies utilize a common set of DNA. The entire mammalian genome is only 3 10 bp. reagents, namely transgenic mouse lines that express the Therefore it is highly unlikely that an exact loxP site is Cre recombinase in a tissue-specific manner and with represented outside of the phage genome. Conse- high fidelity with respect to the recombinase activity. quently, introducing it through transgenesis into the The bottleneck for taking full advantage of these pow- eukaryotic genome restricts the Cre-mediated recombi- erful technologies seems to be the realization of this nation to the exogenous loxP site. The result of the Cre universal set of reagents, the Cre transgenic lines. The purpose of this introductory review in this special * Correspondence to: Andras Nagy, Samuel Lunenfeld Research Institute, issue of Genesis is to summarize the recent status of mouse Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario, M5G 1X5, genetic strategies that apply the Cre recombinase system. Canada. E-mail: [email protected] 100 NAGY FIG. 1. Cre/loxP system. (A) Close-up of Cre recombinase-mediated recombination between two 34 bp loxP sites. Schematics of excision vs. integration (B) and inversion (C). recombinase-mediated recombination depends on the started the development of complex strategies to location and orientation of the loxP sites. They can be achieve far more sophisticated genetic alterations than located cis or trans. In the former situation, their orien- had been possible before. Now we have an enormous set tation can be the same or the opposite (Fig. 1B,C). In the of building blocks from which to select the desired case of trans localization, the DNA strands involved can combinations to achieve particular goals (Nagy, 1996). be linear or circular. The outcome of the recombination As far as imaginable genetic alterations are concerned, between loxP sites depends on the actual scenario con- there is not too much left in the “impossible” category. cerning these variables. It could be excision or inversion Cre recombinase is certainly one of the key tools that of an intervening sequence in the case of cis loxP sites made many of the newly developed genome alteration (Fig. 1B,C) or insertion of one DNA into another or technologies possible. For in vitro recombination be- translocation between two molecules (chromosomes) in tween loxP sites in cis, excision works efficiently with- the case of trans loxP sites. out any active selection if the Cre is transiently ex- ES cell and transgenic technologies quickly recog- pressed from a strong promoter (Nagy et al., 1998). The nized (Gu et al., 1993) the potential in the new tool and range of excision is surprisingly large. We successfully CRE RECOMBINASE 101 excised an almost 400 kb genomic region with 50% sites are needed. Too many loxP sites, however, can lead to efficiency with no selection for the excision event. This a problem of chromosomal instability. It was recently indi- was done using our pCX-NLS-Cre vector (Mar and Nagy, cated that multiple copy integration of loxP sites and the unpublished), in which a Cre recombinase equipped presence of recombinase can lead to chromosome loss with a nuclear localization signal was expressed with the (Lewandoski and Martin, 1997). To stay on the safe side, it exceptionally strong CMV enhancer/chicken ␤-actin pro- is important therefore to ensure that the minimum number moter combination (Niwa et al., 1991). For larger cis of loxP sites is introduced into the cell for a certain strategy. distances or for a trans type of recombination, the fre- The means of introduction of the Cre recombinase quency is smaller, therefore a selection system is re- into the cells bears a much higher number of variations. quired to identify such events in vitro (Ramirez-Solis et The usual in vitro introduction for transient expression al., 1995). The selection is designed in such a way that or stable integration is electroporation or calcium pre- the recombination event is needed to activate the ex- cipitation. Electroporation in fact is surprisingly effi- pression of a positive selectable marker. The most fre- cient. It can reach as high as 70% as measured by the quently applied system is based on the bipartite Hprt Cre-mediated excision rate with the pCX-NLS-Cre vector mini-gene. The two halves of the gene are placed in the (Nagy Lab, unpublished data). The favored method of genome in such a way that the Cre-mediated recombi- introduction of Cre in vivo very much depends on the nation is the only possibility to bring the two pieces application. The simplest solution to achieve overall together and restore the function of the Hprt gene. The excision in the developing embryo is a cross between a only limitation of this strategy is the requirement for an loxP “provider” and a general Cre expresser transgenic Hprt-deficient cell line as the subject of genetic manip- line (Sakai and Miyazaki, 1997; Lallemand et al., 1998). ulation. An alternative solution for a bipartite selectable Another variation of this approach is pronuclear injec- marker is to split the expression construct between the tion of a Cre expression vector, and the resultant tran- promoter and the coding region. We preferred this strategy sient Cre expression does its job during preimplantation in the deletion study mentioned above and proved the development (Araki et al., 1995). Injecting Cre RNA or principle. The third common selection system is based on protein itself may also work. These latter three means the loss of a negative selectable marker (i.e., herpes virus are obviously more tedious than simple mating, but the thymidine kinase; R. Conlon, personal communication). reward is that they do not introduce a Cre transgene into The in vivo Cre-mediated excision is also efficient if the system investigated. The main methodology for lin- the recombinase is expressed at a sufficiently high level eage/cell type-specific excision is the use of established from a transgene. For most of the strategies applying Cre transgenic lines expressing Cre under the control of a recombinase-mediated genome modifications, there is a promoter with the required specificity. In certain appli- requirement for reliable, high-fidelity, and specific trans- cations, however, sporadic expression of Cre is the goal.