A targeting vector was designed to insert a single loxP site upstream of exon 2, and a loxP-flanked neomycin resistance (neo) cassette downstream of exon 3 of the RAP-related protein-1a (Rap1a) gene. The construct was electroporated into 129S/SvEv-derived MM13 embryonic stem (ES) cells. Correctly targeted ES cells were transiently transfected with a cre expression plasmid to delete the neo cassette. Resulting ES cells contained multiple gene rearrangments; intact floxed-exons 2-3, intact floxed-neo cassette, or excision of both exons 2-3 and the neo cassette. Correctly targeted ES cells, containing only the floxed-exons 2-3, were injected into blastocysts and resulting chimeric males were bred with C57BL/6J females.
A second targeting vector was designed to insert a single a loxP-flanked neomycin resistance (neo) cassette upstream of exon 1 and a single loxP site downstream of exon 1 of the RAP-related protein-1b (Rap1b) gene. The construct was electroporated into 129S/SvEv-derived MM13 embryonic stem (ES) cells. Correctly targeted ES cells were transiently transfected with a cre expression plasmid to delete the neo cassette. Resulting ES cells contained multiple gene rearrangments; intact floxed-exon 1, intact floxed-neo cassette, or excision of both exon 1 and the neo cassette. Correctly targeted ES cells, containing only the floxed-exon 1, were injected into blastocysts and resulting chimeric males were bred with C57BL/6J females. Resulting offspring were bred to Rap1a floxed mice, and double floxed mice were maintained on a mixed C57BL/6J;129S background.
Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony.
