Constructs used by Regeneron, Inc. and the CSD (CHORI/Sanger/UC Davis) team
The two groups funded to produce knockout alleles for the KOMP project 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.
Regeneron, Inc. will use the following general construct for creating knockouts for KOMP.
These alleles will be deletions of the major portion of the gene of interest, thus assuring a null knockout.
In particular, this construct is well suited for genes that are small, have a limited number of exons, or show effects with major
deletions that ablate all splice forms.
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.
CSD: CHORI (Children's Hospital Oakland Research Institute), the Wellcome Trust Sanger Institute, and the University of California at Davis
are using two constructs, both of which rely on identifying a key exon relevant to all transcripts of the gene. Each of these constructs can produce reporter
knockouts, conditional knockouts, and null alleles. Thus, this construct is extremely flexible in the types of alleles that can
be produced. However, the genes must be of sufficient size to contain a "differential" exon of note,
and the approach might not be readily amenable for small genes with few exons.
Promoterless Targeting Cassette:
Promoter Driven Targeting Cassette:
See the fuller description of the alleles that
can be produced in the CSD project.
