International Knockout Mouse Consortium

The groups funded to produce IKMC knockout alleles use targeting constructs that have different and complementary properties. Below is a general outline of these constructs that may be helpful in your assessment of the preferred approach for targeting your gene(s) of interest.

Constructs used by KOMP-Regeneron

The VelociGene group at Regeneron, Inc. will use the following general construct for creating knockouts for KOMP. In most cases these will be complete null alleles that delete the entire protein coding sequence of the target gene. This allele design can be applied to any gene transcribed by RNA polymerase II regardless of its size, intron-exon structure, RNA splicing pattern, or protein-coding capacity.

Reference: Valenzuela, D. M., Murphy, A. J., Frendewey, D., Gale, N. W., Economides, A. N., Auerbach, W., Poueymirou, W. T., Adams, N. C., Rojas, J., Yasenchak, J., et al. (2003). High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat Biotechnol 21, 652-659.

Constructs used by KOMP-CSD and EUCOMM

EUCOMM and KOMP-CSD use promoterless and promoter-driven targeting cassettes for the generation of a 'Knockout-first allele' (Testa et al., 2004). 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. Because the target gene must contain a "critical" exon, the approach may not be readily ammenable for small genes with few exons.

"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.

Derivative alleles

tm1a: KO first allele (reporter-tagged insertion allele)

tm1b: post-Cre (reporter-tagged deletion alllele)

tm1c: post-Flp (conditional allele)

tm1d: post-Flp and Cre (deletion allele with no reporter)

tm1e: targeted, non-conditional allele

Reference

Testa G, Schaft J, van der Hoeven F, Glaser S, Anastassiadis K, Zhang Y, Hermann T, Stremmel W, Stewart AF. (2004). A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles. Genesis 38: 151-8.