EUCOMM uses conditional gene targeting and gene trapping strategies for the generation of knockout alleles in C57BL6/N embryonic stem cells.
EUCOMM uses promoterless and promoter-driven targeting cassettes for the generation of a 'Knockout-first allele' (Skarnes et al., 2011) in C57BL/6N embryonic stem cells (Pettitt et al., 2009). This strategy relies on the identification of a 'critical' exon common to all transcript variants that, when deleted, creates a frame-shift mutation. The KO-first allele is flexible and can produce reporter knockouts, conditional knockouts, and null alleles following exposure to site-specific recombinases Cre and Flp. Knockout-first alleles can also be readily modified in cells using dual recombination-mediated cassette exchange (Osterwalder et al., 2010).
"Targeted, non-conditional " alleles (tm1e) are missing the downstream loxP site. The 3’ loxP site is often lost due recombination events in the homology region between the targeting cassette and 3’ loxP site. Approximately one half of the clones retain the loxP site, however, in extreme cases, the loxP site is absent in all clones. These lacZ-tagged alleles report endogenous gene expression and are highly likely to be null mutations. However, these mutations cannot be converted to conditional alleles with Flp recombinase.
tm1a: KO first allele (reporter-tagged insertion allele)
tm1b: Reporter-tagged deletion allele (post-Cre)
tm1c: Conditional allele (post-Flp)
tm1d: Deletion allele (post-Flp and Cre with no reporter)
tm1e: targeted, non-conditional allele
Skarnes WC, Rosen B, West AP, Koutsourakis M, Bushell W, Iyer V, Mujica AO, Thomas M, Harrow J, Cox T, Jackson D, Severin J, Biggs P, Fu J, Nefedov M, de Jong PJ, Stewart AF, Bradley A. (2011). A conditional knockout resource for the genome-wide study of mouse gene function. Nature. 474, 337-342.
Osterwalder M, Galli A, Rosen B, Skarnes WC, Zeller R, Lopez-Rios J. (2010). Dual RMCE for efficient re-engineering of mouse mutant alleles. Nat Methods. 7, 893-895.
Pettitt SJ, Liang Q, Rairdan XY, Moran JL, Prosser HM, Beier DR, Lloyd KC, Bradley A, Skarnes WC. (2009). Agouti C57BL/6N embryonic stem cells for mouse genetic resources. Nat Methods. 6, 493-495.
Gene traps are random insertional mutations in genes expressed in ES cells. The gene trap allele used for EUCOMM is conditional following exposure to site-specific reconbinases Cre and Flp (Schnutgen et al., 2005). The allele can also be modified in cells using recombination mediated cassette exchange (Schebelle et al., 2010).
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. (2005). Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc Natl Acad Sci U S A.102,7221-726.
Schebelle L, Wolf C, Stribl C, Javaheri T, Schnütgen F, Ettinger A, Ivics Z, Hansen J, Ruiz P, von Melchner H, Wurst W, Floss T. (2010). Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps. Nucleic Acids Res. 38, e106.