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An optimized internal ribosome entry sequence (IRES) fused to cre recombinase followed by a neomycin resistance cassette flanked FRT sites was inserted after the Ch AT stop codon in BAC (b MQ185k21) using homologous recombination in bacteria.

A genomic Ch AT fragment with 4kb homology arms on either side of the IRES-Cre insertion site was lifted out from the BAC using reverse recombineering techniques and transformed into ES cells.

The construct was electroporated into 129S6/Sv Ev Tac-derived W4 embryonic stem (ES) cells.

Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting mutant offspring (still retaining the frt-flanked neo) were bred to wildtype siblings, and maintained as such prior to sending heterozygous males to The Jackson Laboratory Repository in 2006.

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