A targeting vector was designed to replace exon 2 and a portion of exon 3 (collectively encoding IgV and part of a transmembrane domain) of the cytotoxic T-lymphocyte-associated protein 4 gene (Ctla4) on chromosome 1 with a PGK-neo cassette. This construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells. ES cell clone T5 was found to be heterozygous for the CTLA4 mutation and was able to gave rise to CTLA4-deficient mice exhibiting a lymphoproliferative phenotype. Because of the close proximity of Ctla4 and the CD28 antigen gene (Cd28) on chromosome 1, T5 ES cells were later re-targeted with a vector designed to replace part of the IgV-like exon 2 and a portion of transmembrane exon 3 of Cd28 with a PGK-hygro cassette. Correctly targeted ES cells bearing both mutations were injected into recipient blastocysts. The resulting chimeric animals were bred to C57BL/6J wildtype mice to generate the double mutant colony. Because of their close proximity, Ctla4 Cd28 double mutant mice were bred together by selecting progeny in which the two mutations co-segregated (indicating both mutations were on the same heterologous chromosome). Next, double mutant mice were backcrossed to C57BL/6 wildtype mice for at least ten generations (see SNP results below). In 2013, males homozygous for both knockout mutations (CD28/CTLA-4-/-) were sent to The Jackson Laboratory Repository and used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the first generation rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 2 of 5 markers that determine C57BL/6J from C57BL/6N (one each on chromosome 8 and 19) were found to be heterozygous. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J;C57BL/6N genetic background.
