The pMADMα 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 (tdT3Myc^ATG-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). To convert pMADMα to the "MADM GT" or "MADM TG" genotype, ES cells were transfected with a FLP-expressing plasmid.

The resulting ES cells with recombination between the first and second frt sites (retaining the "MADM TG" cassette and deleting the "MADM GT" cassette) were microinjected into recipient blastocysts.
Chimeric progeny were bred to either CD1 or C57BL/6J mice. The resulting Miya10^TG (M10^TG) mice were bred to mice with germline Cre recombinase expression to remove the floxed neo cassette. M10^TG mice were maintained on a mixed genetic background (mixed CD1, 129, C57BL/6 and/or FVB/N) for several generations (and the Cre recombinase gene removed) prior to sending to The Jackson Laboratory Repository. Upon arrival, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish The Jackson Laboratory Repository colony.

• Noncarrier

Additional Information

• Considerations for Choosing Controls