Knock-in mice were created using embryonic stem cells and Cre/lox P technology. A 13 kb genomic clone containing the relevant segment of the murine Col1a2 gene was isolated from a λ phage library constructed with gDNA of the 129Sv/Ev Taconic mouse strain. PCR site-directed mutagenesis was used to change the targeted codon from GGT to TGT. The Col1a2 G610C-positive embryonic stem cells were injected into blastocysts of C57BL/6J (B6) mice. Founder mice that retain the neo targeting vector are termed neo+ or G610C Neo mice. Progeny obtained by breeding a founder male with a female that expressed Cre recombinase (Jackson Laboratory, Bar Harbor, ME, USA, Stock Number 003724) are termed neo– or G610C OI mice. The G610C OI mouse line is available to the research community through the Jackson Laboratory Mouse Repository (Jax Stock Number: 007248).
Four F1 strains of G610C OI mice were used to determine the effect of genetic background on phenotype in 2-month-old male mice. Incipient congenic G610C OI B6 (∼98% B6 genetic background) male breeders were generated by six generations of backcrosses to Jackson Laboratory B6 mice (Stock Number 000664). Experimental heterozygous B6 male breeders were crossed with A/J (Stock Number 000646), BALB/cByJ (Stock Number 001026), C3H/HeJ (Stock Number 000659), and FVB/NJ (Stock Number 001806) females purchased from the Jackson Laboratory. Progeny of these crosses are designated, respectively, as A.B6, Cby.B6, C3.B6, and FVB.B6. Experimental mice were housed at UMB in a single specific-pathogen-free room and were exposed to identical environmental conditions consisting of a 12 hour light/dark cycle, an ambient temperature of 23°C, and ad libitum access to water and laboratory mouse chow. Genotype was assigned using a PCR assay that can discriminate the three possible G610C OI mouse genotypes. The forward primer (TCC CTG CTT GCC CTA GTC CCA AAG ATC CTT) and the reverse primer (AAG GTA TAG ATC AGA CAG CTG GCA CAT CCA) will generate a 165 bp (wild type) or a 337 and a 165 bp (heterozygous) or a 337 bp (homozygous) PCR product using G610C OI mice gDNA. All animals were euthanized by CO2 asphyxiation.
Four F1 strains of G610C OI mice were used to determine the effect of genetic background on phenotype in 2-month-old male mice. Incipient congenic G610C OI B6 (∼98% B6 genetic background) male breeders were generated by six generations of backcrosses to Jackson Laboratory B6 mice (Stock Number 000664). Experimental heterozygous B6 male breeders were crossed with A/J (Stock Number 000646), BALB/cByJ (Stock Number 001026), C3H/HeJ (Stock Number 000659), and FVB/NJ (Stock Number 001806) females purchased from the Jackson Laboratory. Progeny of these crosses are designated, respectively, as A.B6, Cby.B6, C3.B6, and FVB.B6. Experimental mice were housed at UMB in a single specific-pathogen-free room and were exposed to identical environmental conditions consisting of a 12 hour light/dark cycle, an ambient temperature of 23°C, and ad libitum access to water and laboratory mouse chow. Genotype was assigned using a PCR assay that can discriminate the three possible G610C OI mouse genotypes. The forward primer (TCC CTG CTT GCC CTA GTC CCA AAG ATC CTT) and the reverse primer (AAG GTA TAG ATC AGA CAG CTG GCA CAT CCA) will generate a 165 bp (wild type) or a 337 and a 165 bp (heterozygous) or a 337 bp (homozygous) PCR product using G610C OI mice gDNA. All animals were euthanized by CO2 asphyxiation.
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