A targeting vector was designed to replace exons 5-7 of the proteasome (prosome, macropain) subunit, beta type 10 (Psmb10) gene with a neomycin resistance (neo) cassette. The construct was electroporated into 129S2/SvPas-derived D3 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric males were bred to C57BL/6 females.
Another targeting vector was designed to replace exons 1-5 of the proteasome (prosome, macropain) subunit, beta type 8 (Psmb8) gene with a neomycin resistance (neo) cassette. The construct was electroporated into 129P2/OlaHsd-derived E14.1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric males were bred to C57BL/6 females to generate a colony of Psmb8tm1Hjf knockout mice.
Mice carrying the mutations were crossed, and the resulting double mutants were backcrossed for at least 10 generations to C57BL/6 mice by the donating lab (see SNP note below). Upon arrival at The Jackson Laboratory, mice were bred to C57BL/6J (Stock No. 000664) for at least one generation to establish the colony. The Psmb8tm1Hjf allele was selected against, and was maintained as a separate strain (Stock No. 021201).
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 rederived living colony at The Jackson Laboratory Repository. Five of the 27 markers throughout the genome were segregating, suggested an incomplete backcross. One marker on Chromosome 11 was segregating from an unknown source.
