These double mutant mice, heterozygous for the targeted mutation and hemizygous for the transgene, were generated by crossing transgenic mice (founder line YG8) with Fxntm1Mkn targeted mutant mice.
Drs. Michel Koenig and Helene Puccio generated the Fxn targeted mutation allele by disrupting exon 4 and flanking sequences with a loxP site flanked PGK-neo cassette. The construct was electroporated into 129/Sv derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts. The resulting chimeric animals were crossed to C57BL/6J mice, and then backcrossed to C57BL/6J for five generations.
The transgenic construct consisting of a 370kb YAC insert that includes the entire human FXN gene with GAA triplet repeat sequences, was injected into fertilized CBA X C57BL/6 hybrid fertilized oocytes. Founder line YG8 was established, carrying 2 tandem copies of the human FXN gene with GAA trinucleotide sequence repeats as low as 122 and as high as 207. Founder animals were bred to wildtype C57BL/6J mice for five generations.
The two lines were then crossed to generate the double mutant. The Donating Investigator breeds mice that are heterozygous for the Fxntm1Mkn targeted mutation and hemizygous for the YG8 transgene to mice that are heterozygous for the Fxntm1Mkn targeted mutation to obtain mice homozygous for the Fxntm1Mkn targeted mutation and hemizygous for the YG8 transgene.
SNP (single nucleotide polymorphism) analysis performed by The Jackson Laboratory revealed that the mice originally imported were on a mixed genetic background. The mixed genetic background mice were then backcrossed to C57BL/6J for five generations.
