For the Park7 allele, a targeting vector designed by Dr. Jie Shen (Brigham
& Women's Hospital, Harvard University) containing a PGK-neomycin cassette was used to disrupt exon 2. The construct was electroporated into (C57BL/6 x 129)F1 derived MKV6.5 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission.
The mice were backcrossed to C57BL/6J for 10 generations.
For the Gpx1tm1Ysh allele, a targeting vector designed by Dr. Ye-Shih Ho (Wayne State University) containing a NEO cassette was used to disrupt exon 2. The construct was electroporated into 129 derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts. The resulting chimeric animals were tested for germline transmission.
The mice were crossed for approximately 6 generations to C57BL/6. Dr. Matthew Goldberg obtained the mice and backcrossed them to C57BL/6J for 3 or 4 generations. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6 genetic background.
For the Park2 allele, a targeting vector designed by a targeting vector designed by Dr. Jie Shen (Brigham & Women's Hospital, Harvard University) containing an in-frame EGFP coding sequence (followed by translation and transcription termination sequences and a PGK-neomycin cassette) was used to disrupt most of exon 3. The construct was electroporated into the 129S4/SvJae-derived J1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were backcrossed to C57BL/6J for 10 generations.
To generate this triple mutant strain, the Park7 and Park2 knockout lines were bred and intercrossed to obtain double knockout mice. The double knockout mice were then crossed to the Gpx1 knockout mice for 2 generations, and then intercrossed to obtain mice homozygous for all 3 alleles, Parkin-/- DJ-1-/- Gpx1-/- (See SNP note below). Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While 26 of the 27 markers throughout the genome suggested a C57BL/6 genetic background, 2 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.
