129/SvEv-derived male embryonic stem cells (ES) were used to target the Ndufa1S55A allele on its native locus at X-chromosome. The Ndufa1S55A allele harbors a point mutation in the codon-55 (T>G; codon TCT>GCT). The long homology arm was 6.0 kb upstream to the point mutation (T>G) in exon 2. The LoxP/FRT-Neo cassette for G418 selection was inserted 903 bp downstream of the point mutation within intron 2-3. The short homology arm extended 2.0 kb from the 5’-end of the Neo cassette. The T>G mutation was created by site-directed mutagenesis. ES clones with correct targeting by homologous recombination were screened first by polymerase chain reaction (PCR) and later confirmed by Southern blot analysis with an internal probe. Genomic DNA was digested with Hpa
I, and electrophoretically separated on a 0.8% agarose gel. After transfer to a nylon membrane, the digested DNA was hybridized with a probe targeted against the 3’ internal region. DNA from 129/SvEv was used as wild-type control. The expected sizes for wild-type (WT; 9.3 kb) and knock-in (KI Neo: 11 kb) were observed. Because ES cells were derived from male mice, only one copy of the Ndufa1
gene was expected. Therefore, correct targeting will result in only one band of 11 kb. By Southern blot analysis clone #253 was identified to have Ndufa1S55A allele correctly targeted at its native locus. After confirming the reduced MWFE and Complex I levels in ES clone #253, it was injected into C57BL/6 blastocysts.
Chimeras with high percentage agouti coat color were mated to 129 FLP mice (JAX #003946
) to remove the Neo-cassette and simultaneously generate the germ-line founder mice. A mutant male (Ndufa1S55A/Y) and two heterozygous females (Ndufa1S55A/+) were obtained. They were crossed with wild-type mice (129S1/SvImJ, JAX #02448
) to expand the colony. The deletion of Neo-cassette was confirmed by PCR screening using primers set F1 (5’-CAA AGC CAT TTC TCT GCC TCT-3’) and R1 (5’-CCA AAA AAC CTC ACT CAC AAC C-3’). The presence of Ndufa1S55A allele was confirmed using primers set F2 (5’ -TGA GCT ATC TCT TCA GCC TCT AG- 3’) and R2 (5’-GAC CCT GTC TCA ATT ACA AAT CAA CCA ACC -3’) by Hyp188
III restriction fragment length polymorphism. The enzyme cuts only the wild-type (Ndufa1+) allele. Finally, the presence of T>G mutation in the Ndufa1S55A mice was confirmed by DNA sequencing. From gene targeting to founder mice generation, this project was supported by inGenious Targeting Laboratory (Stony Brook, NY) on a pay-for-service basis.
To transfer the Ndufa1S55A allele on BALB/c genetic background, we performed 9 generations of backcrossing. Because the Ndufa1 gene is X-chromosome linked, we crossed Ndufa1S55A-129S1 mutant males (r/Y) mice (from N5) with wild-type BALB/c females (X/X). Such a cross resulted in heterozygous females (r/X), which were mated with wild-type BALB/c males (X/Y). Then in the next generation, mutant male progeny (r/Y) were mated with wild-type BALB/c females (X/X). This alternating breeding scheme continued for another 8 generations (i.e., N9) to generate Ndufa1S55A-BALB/c strain.