The pMADMalpha transgene was designed with the CMV enhancer/chicken beta-actin core promoter (pCA) promoter, a frt site, the MADM GT cassette, and second frt site, the MADM TG cassette, a third frt site, and an SV40 T-antigen poly(A) signal. The MADM GT cassette used here has the N-terminal portion of a mutant enhanced green fluorescent protein (mut4EGFP first exon; nucleotides 1-274), a beta-globin intronic sequence (containing a loxP-flanked neomycin resistance gene), and ATG-less tdTomato sequence (nucleotides 3-1548 including stop codon) tagged with three copies of the Myc epitope at its C-terminus (tdT3MycATG-less). The MADM TG cassette used here has an ATG start codon, a beta-globin intronic sequence (containing a loxP-flanked neomycin resistance gene), and the C-terminal portion of a mutant enhanced green fluorescent protein (mut4EGFP second exon; nucleotides 275-735 including stop codon). The transgene was electroporated into (129X1/SvJ x 129S1/Sv)F1-derived R1 embryonic stem (ES) cells. ES cells were screened for intact single-copy transgenes with integration in intergenic regions close to any centromere. ES cells were identified with an intact single-copy of the transgene inserted into an intergenic region at ~21 Mbp on chromosome 10 between the Ahi1 and Myb loci; this new insertion locus was named Miya10 (or M10). The transgene inserted into an intergenic region at ~21 Mbp on chromosome 10 between the Ahi1 and Myb loci; this new insertion locus was named Miya10 (or M10). ]Cre-mediated recombination removed the neo cassette. (J:181830)
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