EUCOMMTOOLs uses gene targeting and gene knock-in strategies for the generation of conditional knockout alleles and CreERT2 knock-in alleles, respectively, in C57BL6/N embryonic stem cells (Pettitt et al., 2009).
EUCOMMTOOLs uses promoterless and promoter-driven targeting cassettes for the generation of a 'Knockout-first allele' (Skarnes et al., 2011). 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).
In cases where the target gene is small and does not contain a "critical" exon, the approach is to split an exon and introduce the targeting cassette imbedded in an ‘artificial intron’.
"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.
EUCOMMTOOLs uses targeting vectors that contain green fluorescent protein and Cre-ERT2 separated with a 2A peptide sequence. The selection cassette is flanked with Rox sites and is removed using Dre recombinase.