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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Genesis. Author manuscript; available in PMC Oct 1, 2011.
Published in final edited form as:
PMCID: PMC2958239
NIHMSID: NIHMS231923

ROSA26Flpo deleter mice promote efficient inversion of conditional gene traps in vivo

Abstract

Gene trap mutagenesis in embryonic stem (ES) cells is an important tool to help elucidate gene function in current mouse mutagenesis efforts. Vector systems based on inversion of the gene trap module have recently been devised to allow for conditional mutagenesis. However, additional efforts are needed to improve this technology including improving the efficiency of site-specific recombinases required to manipulate these conditional vectors in vivo. Here we describe a mouse line carrying the codon-optimized FLP recombinase Flpo at the ROSA26 locus that functions at higher efficiency than a similar Flpe line in mediating the DNA inversion of a conditional gene trap cassette in vivo.

Keywords: Mouse, mutagenesis, gene trap, FLP, site-specific recombinase, ES cells

Introduction

In the post-genomic era, attention has focused on the functional annotation of individual genes in the mouse. Large-scale mutagenesis efforts by KOMP, EUCOMM, NorCOMM, and the International Gene Trap Consortium are employing a number of techniques such as conditional and non-conditional gene targeting strategies, as well as gene trapping techniques in mouse embryonic stem (ES) cells to generate mutant alleles (Austin et al., 2004; Friedel et al., 2007; Skarnes et al., 2004).

Gene trapping is an insertional mutagenesis technique that is designed to mutate a gene with a trap cassette that monitors its expression and serves as a molecular tag for its identification. Typically, a retroviral or plasmid gene trap vector contains a splice acceptor (SA) sequence followed by a promoter-less selection/reporter cassette and a transcription terminating polyadenylation (pA) sequence [for a review, see (Stanford et al., 2001)]. One limitation of the gene trap methodology is that most commonly used vectors do not allow for conditional mutagenesis. Since approximately 25% of trapped genes are required for embryonic development (Friedrich and Soriano, 1991; Mitchell et al., 2001), embryonic lethality precludes the study of these genes in the adult mouse, or their function in specific tissues. Recently, conditional gene trapping vector systems, such as the FlEx vectors, have been described and are currently being employed by EUCOMM to generate mutant alleles in mouse ES cells (Schnütgen et al., 2005). The FlEx vectors feature a gene trap cassette that is flanked by heterotypic recognition sequences in a head to head orientation for two site-specific DNA recombinases (SSRs). The FlEx system relies on sequential use of the Cre and FLP SSRs to conditionally inactivate and reactivate the gene traps. Upon recombination with the first SSR, the gene trap cassette is reversed from a mutagenic direct orientation (DO) to a non-mutagenic reverse orientation (RO). Subsequent use of a second SSR leads to an inversion of the gene trap back to the original mutagenic direct orientation. If this is done in mice, mutagenesis can be conducted in a tissue-specific manner, thus circumventing the potential embryonic lethality.

Both the P1 bacteriophage-derived Cre and the yeast derived FLP have been used in mammals to mediate DNA recombination, including deletions, insertions and inversions (Buchholz et al., 1998; Dymecki, 1996; O’Gorman et al., 1991; Sauer and Henderson, 1988). We have recently shown that a codon-optimized FLP recombinase (Flpo) mediates DNA recombination in ES cells more efficiently than the currently used variant Flpe (Raymond and Soriano, 2007). Here we describe a mouse strain that ubiquitously expresses Flpo and we show that this optimized SSR can be used to efficiently mediate the DNA inversion of a FlEx conditional gene trap cassette.

Results

We had previously shown that a codon-optimized Flpo gene improved the recombinase efficiency in vitro in mouse embryonic stem (ES) cells (Raymond and Soriano, 2007). To test the in vivo performance of the optimized recombinase, we targeted the Flpo gene into the broadly-expressed ROSA26 locus (Soriano, 1999) and subsequently generated heterozygous and homozygous ROSA26Flpo mice (Figure 1). These mice were maintained on a 129S4 co-isogenic background, were fertile and did not exhibit any overt phenotype, similar to mice constitutively expressing the Flpe gene from the ROSA26 locus (Farley et al., 2000).

Figure 1
Strategy for conditional gene trap inactivation

To assess the recombination efficiency of the Flpo gene compared to the Flpe and Cre recombinases in vivo, we generated a gene trap mouse line harboring the FlEx conditional gene trap vector, FlipROSAβgeo*, as this vector is responsive to inversions by both Cre and FLP recombinase (Schnütgen et al., 2005) (Figure 2A). We first tested that the functionality of the FlEx conditional gene trap vector by mating mice heterozygous for the FlipROSAβgeo* allele to Meox2Cre (MORE) heterozygous mice. MORE mice express Cre from the Meox2 locus leading to generalized recombinase activity in the mouse epiblast but not in the extra-embryonic tissues (Tallquist and Soriano, 2000). As the gene trap mice only exhibited diffuse lacZ staining, we assessed inversion efficiency by Southern blotting. This analysis on genomic DNA from tail biopsies of the resultant pups from this cross yielded 2 animals harboring both the FlipROSAβgeo* and the MORE allele. Both of these mice displayed successful recombinase-mediated inversion of the gene trap allele, with some lack of inversion due to the mosaic expression of Cre recombinase in this strain (Hayashi et al., 2002)(Figure 2Ba).

Figure 2
Comparison of the efficiency of 3 recombinases in mediating DNA inversions in vivo

Next we tested the ability of Flpe driven from the ROSA26 locus (FlpeR) to mediate the inversion of the gene trap allele. Both of the mice that were positive for the Flpe and the gene trap allele only displayed a low level of recombinase mediated inversion to the non-mutagenic reverse orientation, while the majority remained in the forward mutagenic orientation (Figure 2Bb). Last, we tested the recombination efficiency of the optimized Flpo recombinase in vivo by mating heterozygous ROSA26Flpo mice to mice heterozygous for the FlipROSAβgeo* gene trap allele. Animals positive for both the Flpo recombinase and gene trap alleles displayed a complete inversion of the FlipROSAβgeo* cassette to the non-mutagenic reverse orientation (Figure 2Bc). As expected, these mice have also been shown to be efficient for mediating deletion of sequences flanked by FRT sites in the same orientation (not shown). These results indicate that the optimized Flpo gene is functional in vivo and displays enhanced recombination activity in mediating DNA inversions compared to Flpe when expressed from the ROSA26 locus.

Discussion

In this report, we show that a mouse codon-optimized Flpo gene displayed enhanced efficiency in mediating DNA recombination in mice compared to Flpe, as it does in ES cells. Our results are consistent with two recent reports that have shown increased efficiency of Flpo relative to Flpe in mice for mediating deletions of sequences flanked by FRT sites in the same orientation (Kranz et al., 2010; Wu et al., 2009). However comparison of Flpo and Flpe efficiencies was complicated by the use of transgenic lines with random insertions rather than knock-ins as well as differing promoters. We now demonstrate by direct comparison that Flpo also exhibits enhanced activity compared to Flpe in vivo. The ROSA26Flpo mice were efficient in mediating the inversion of a FlEx conditional gene trap vector. As large ES cell libraries of these conditional gene traps are currently being constructed by the European Conditional Mouse Mutagenesis (EUCOMM) initiative, this new mouse strain will prove to be an invaluable resource in fully-enabling this technology. Furthermore, tissue-specific Flpo or Flpo-ER lines may provide in future a set of tools that complements the existing resource of tissue-specific Cre lines. The ROSA26Flpo mouse strain can be obtained from the Jackson Laboratory (JR7844) and will be made freely available to the academic scientific community.

Materials and Methods

Recombinase and conditional gene trap constructs and mice

The optimized Flpo coding sequence was blunt cloned into the pROSA26-1 vector to generate mouse strains that broadly express this SSR from the ROSA26 locus as previously described (Raymond and Soriano, 2007). Consistent to the recommendations of the International Committee on Standardized Genetic Nomenclature for Mice, the strain name is: 129S4-Gt(ROSA)26Sortm2(FLPo)Sor/J. The Meox2Cre and Flper mouse strains were previously described (Farley et al., 2000; Tallquist and Soriano, 2000).

The conditional gene trap vector pFlipROSAβgeo* was a kind gift from F. Schnütgen (Schnütgen et al., 2005). Gene trap retrovirus was produced from the pFlipROSAβgeo* plasmid in GP+E86 helper cells, and AK7 ES cells were infected with the viral-containing supernatant at a multiplicity of infection of 0.1 as described previously (Soriano et al., 1991). G418-resistant ES cell lines were subsequently used to generate conditional gene trap mouse lines to be used for analysis.

Southern Blotting

Genomic DNA was isolated from mouse tail biopsies and digested with NcoI. Southern blot analysis was performed with a neomycin probe to determine the direct- or reverse-orientation of the FlipROSAβgeo* conditional gene trap cassette.

Acknowledgments

We thank members of the Soriano laboratory for critical comments on the manuscript and Philip Corrin for technical support. Work in the Soriano laboratory was supported by RO1HD24875 and R37HD25326 from the National Institutes of Child Health and Human Development.

Literature Cited

  • Austin CP, Battey JF, Bradley A, Bucan M, Capecchi M, Collins FS, Dove WF, Duyk G, Dymecki S, Eppig JT, Grieder FB, Heintz N, Hicks G, Insel TR, Joyner A, Koller BH, Lloyd KC, Magnuson T, Moore MW, Nagy A, Pollock JD, Roses AD, Sands AT, Seed B, Skarnes WC, Snoddy J, Soriano P, Stewart DJ, Stewart F, Stillman B, Varmus H, Varticovski L, Verma IM, Vogt TF, von Melchner H, Witkowski J, Woychik RP, Wurst W, Yancopoulos GD, Young SG, Zambrowicz B. The knockout mouse project. Nat Genet. 2004;36:921–924. [PMC free article] [PubMed]
  • Buchholz F, Angrand PO, Stewart AF. Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol. 1998;16:657–662. [PubMed]
  • Dymecki SM. Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. Proc Natl Acad Sci U S A. 1996;93:6191–6196. [PMC free article] [PubMed]
  • Farley FW, Soriano P, Steffen LS, Dymecki SM. Widespread recombinase expression using FLPeR (flipper) mice. Genesis. 2000;28:106–110. [PubMed]
  • Friedel RH, Seisenberger C, Kaloff C, Wurst W. EUCOMM--the European conditional mouse mutagenesis program. Brief Funct Genomic Proteomic. 2007;6:180–185. [PubMed]
  • Friedrich G, Soriano P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 1991;5:1513–1523. [PubMed]
  • Hayashi S, Lewis P, Pevny L, McMahon AP. Efficient gene modulation in mouse epiblast using a Sox2Cre transgenic mouse strain. Gene Expr Patterns. 2002;2:93–97. [PubMed]
  • Kranz A, Fu J, Duerschke K, Weidlich S, Naumann R, Stewart AF, Anastassiadis K. An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase. Genesis 2010 [PubMed]
  • Mitchell KJ, Pinson KI, Kelly OG, Brennan J, Zupicich J, Scherz P, Leighton PA, Goodrich LV, Lu X, Avery BJ, Tate P, Dill K, Pangilinan E, Wakenight P, Tessier-Lavigne M, Skarnes WC. Functional analysis of secreted and transmembrane proteins critical to mouse development. Nat Genet. 2001;28:241–249. [PubMed]
  • O’Gorman S, Fox DT, Wahl GM. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science. 1991;251:1351–1355. [PubMed]
  • Raymond CS, Soriano P. High-efficiency FLP and PhiC31 site-specific recombination in mammalian cells. PLoS One. 2007;2:e162. [PMC free article] [PubMed]
  • Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A. 1988;85:5166–5170. [PMC free article] [PubMed]
  • Schnütgen F, De-Zolt S, Van Sloun P, Hollatz M, Floss T, Hansen J, Altschmied J, Seisenberger C, Ghyselinck NB, Ruiz P, Chambon P, Wurst W, von Melchner H. Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc Natl Acad Sci U S A. 2005;102:7221–7226. [PMC free article] [PubMed]
  • Skarnes WC, von Melchner H, Wurst W, Hicks G, Nord AS, Cox T, Young SG, Ruiz P, Soriano P, Tessier-Lavigne M, Conklin BR, Stanford WL, Rossant J. A public gene trap resource for mouse functional genomics. Nat Genet. 2004;36:543–544. [PMC free article] [PubMed]
  • Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet. 1999;21:70–71. [PubMed]
  • Soriano P, Friedrich G, Lawinger P. Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells. J Virol. 1991;65:2314–2319. [PMC free article] [PubMed]
  • Stanford WL, Cohn JB, Cordes SP. Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet. 2001;2:756–768. [PubMed]
  • Tallquist MD, Soriano P. Epiblast-restricted Cre expression in MORE mice: a tool to distinguish embryonic vs. extra-embryonic gene function. Genesis. 2000;26:113–115. [PubMed]
  • Wu Y, Wang C, Sun H, LeRoith D, Yakar S. High-efficient FLPo deleter mice in C57BL/6J background. PLoS One. 2009;4:e8054. [PMC free article] [PubMed]
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