All the mutant lines analysed have been generated at the Wellcome Trust Sanger Institute, using the highly characterised inbred strain C57BL/6. Typically the genetic background is derived from a cross of the C57BL/6 sub-strains C57BL/6N and C57BL/6NTac; the specific strain of each embryo is listed within the embryo details sections on individual embryo pages.
Knockout lines are primarily generated using Embryonic Stem (ES) cell injection. ES cells are targeted with a tm1a knockout first allele, then injected into mouse blastocysts. The tm1a allele is flexible and can produce reporter knockouts, conditional knockouts, and null alleles following exposure to the site-specific recombinases Cre and Flp.
It is well-known that the neo selection cassette can affect the expression of neighbouring genes and the resulting phenotypes observed. For this reason, the selection cassette is removed by Cre excision, producing a tm1b allele that is used for phenotyping.
The tm1a allele is banked with the European Mouse Mutant Archive (EMMA), and can be ordered from EMMA for further study. The construct of the tm1a allele means that it can be used to generate either full or conditional knockouts. These are further described in Skarnes et al, 2011, referenced in Publications.
A small number of DMDD alleles are tm1e, a targeted non-conditional allele. These are identical to the tm1a, except for a missing loxP site downstream of the critical exon caused by a recombination event. For these lines, the tm1e allele is also banked with EMMA. Targeted non-conditional alleles are highly likely to be null, but cannot be converted to conditional alleles with Flp recombinase. Further information on allele nomenclature.
The advent of the CRISPR-Cas9 technique has significantly reduced the time required to generate knockout mouse lines. As a result, some DMDD lines have been produced using this method.
Following allele generation, heterozygote knockout mice are bred together, with a litter of pups expected in the ratio ¼ wild-type, ½ heterozygote and ¼ homozygote. If no homozygous pups are observed at P14, the line is embryonic lethal. If fewer than 13% homozygous pups are observed at P14, the line is classed as sub-viable.
Both embryonic lethal and sub-viable lines enter the DMDD pipeline.