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Targeting and Generation of MCAD-Deficient Mice\nMCAD insertion vector (MCAD IV2) was designed to undergo gap repair of the 1.3-kb deleted region upon homologous recombination in 129P2 (129P2/OlaHsd) ES cells E14–1. Correct targeting of the MCAD locus resulted in a duplication of exons 8, 9, and 10 and integration of flanking plasmid and Neo sequences (Figure 1A). The insertion vector was designed to duplicate exon 8, 9, and 10 at the MCAD locus. Translation of the duplicated exon 8 region results in the formation of premature stop codons resulting in truncation of the MCAD monomer. Specifically, the first premature stop codon arises after translation of only seven amino acids from the duplicated exon 8. The resulting MCAD monomer is missing the C-terminal domain α-helixes that are responsible for making intersubunit contacts to generate the functional MCAD homotetramer.\nFigure 1 Strategy for Disruption of the Mouse Acadm Gene\n(A) The MCAD IV2 insertion targeting vector with a deleted 1.3-kb region encompassing exon 10 and flanking sequences. MCAD IV2 undergoes gap repair upon homologous recombination at the endogenous Acadm locus resulting in a duplication of exons 8, 9, and 10 at the disrupted allele.\n(B) Southern blot analysis of EcoRI-digested genomic DNA from ES cells screened by PCR. Probe A, a DNA fragment consisting of a portion of exon 10 that is not present in the targeting vector, hybridizes to an endogenous 3.1-kb fragment and, upon homologous recombination, to a 13.2-kb fragment. Lane 1 represents a wild-type ES cell line, and Lane 2 and 3 represent targeted ES cell lines. ES cell clones were screened by PCR (data not shown) and confirmed by Southern blot analysis. Southern blot analysis used an exon 10 probe (probe A), not present in the targeting vector, hybridized to a 13.2-kb band in addition to the 3.1-kb endogenous band indicating targeted insertion of the vector at the Acadm locus (Figure 1B). Correctly targeted ES cell clones were microinjected into B6 (C57BL/6NTac) blastocysts to generate chimeric mice. Chimeric mice were backcrossed to both 129P2 and B6 inbred mice to produce MCAD+/− and eventually MCAD−/− mice on a B6/129 mixed background. The studies described here were conducted exclusively on the B6/129 mixed background compared with littermate controls or B6/129 control groups maintained by intercrosses as were the mutants. Perpetuating this mutation as a congenic mutant line on the 129P2 background proved impractical. The 129P2 mice were poor breeders as wild-types, and when introduced, the Acadm mutation was nearly lost on this background because of the high rate of neonatal death. Because of the molecular structure of the targeted allele, it proved virtually impossible to distinguish all three potential genotypes. We could clearly detect the presence or absence of the targeted allele, however, whether a particular mouse was MCAD−/− or MCAD+/− could not be determined by Southern blot or PCR of genomic DNA. Ultimately MCAD−/− mice were ascertained by immunoblot analysis of offspring with subsequent perpetuation of MCAD−/− and MCAD+/+ mice as separate groups."}

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Correct targeting of the MCAD locus resulted in a duplication of exons 8, 9, and 10 and integration of flanking plasmid and Neo sequences (Figure 1A). The insertion vector was designed to duplicate exon 8, 9, and 10 at the MCAD locus. Translation of the duplicated exon 8 region results in the formation of premature stop codons resulting in truncation of the MCAD monomer. Specifically, the first premature stop codon arises after translation of only seven amino acids from the duplicated exon 8. The resulting MCAD monomer is missing the C-terminal domain α-helixes that are responsible for making intersubunit contacts to generate the functional MCAD homotetramer.\nFigure 1 Strategy for Disruption of the Mouse Acadm Gene\n(A) The MCAD IV2 insertion targeting vector with a deleted 1.3-kb region encompassing exon 10 and flanking sequences. MCAD IV2 undergoes gap repair upon homologous recombination at the endogenous Acadm locus resulting in a duplication of exons 8, 9, and 10 at the disrupted allele.\n(B) Southern blot analysis of EcoRI-digested genomic DNA from ES cells screened by PCR. Probe A, a DNA fragment consisting of a portion of exon 10 that is not present in the targeting vector, hybridizes to an endogenous 3.1-kb fragment and, upon homologous recombination, to a 13.2-kb fragment. Lane 1 represents a wild-type ES cell line, and Lane 2 and 3 represent targeted ES cell lines. ES cell clones were screened by PCR (data not shown) and confirmed by Southern blot analysis. Southern blot analysis used an exon 10 probe (probe A), not present in the targeting vector, hybridized to a 13.2-kb band in addition to the 3.1-kb endogenous band indicating targeted insertion of the vector at the Acadm locus (Figure 1B). Correctly targeted ES cell clones were microinjected into B6 (C57BL/6NTac) blastocysts to generate chimeric mice. Chimeric mice were backcrossed to both 129P2 and B6 inbred mice to produce MCAD+/− and eventually MCAD−/− mice on a B6/129 mixed background. The studies described here were conducted exclusively on the B6/129 mixed background compared with littermate controls or B6/129 control groups maintained by intercrosses as were the mutants. Perpetuating this mutation as a congenic mutant line on the 129P2 background proved impractical. The 129P2 mice were poor breeders as wild-types, and when introduced, the Acadm mutation was nearly lost on this background because of the high rate of neonatal death. Because of the molecular structure of the targeted allele, it proved virtually impossible to distinguish all three potential genotypes. We could clearly detect the presence or absence of the targeted allele, however, whether a particular mouse was MCAD−/− or MCAD+/− could not be determined by Southern blot or PCR of genomic DNA. Ultimately MCAD−/− mice were ascertained by immunoblot analysis of offspring with subsequent perpetuation of MCAD−/− and MCAD+/+ mice as separate groups."}

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Targeting and Generation of MCAD-Deficient Mice\nMCAD insertion vector (MCAD IV2) was designed to undergo gap repair of the 1.3-kb deleted region upon homologous recombination in 129P2 (129P2/OlaHsd) ES cells E14–1. Correct targeting of the MCAD locus resulted in a duplication of exons 8, 9, and 10 and integration of flanking plasmid and Neo sequences (Figure 1A). The insertion vector was designed to duplicate exon 8, 9, and 10 at the MCAD locus. Translation of the duplicated exon 8 region results in the formation of premature stop codons resulting in truncation of the MCAD monomer. Specifically, the first premature stop codon arises after translation of only seven amino acids from the duplicated exon 8. The resulting MCAD monomer is missing the C-terminal domain α-helixes that are responsible for making intersubunit contacts to generate the functional MCAD homotetramer.\nFigure 1 Strategy for Disruption of the Mouse Acadm Gene\n(A) The MCAD IV2 insertion targeting vector with a deleted 1.3-kb region encompassing exon 10 and flanking sequences. MCAD IV2 undergoes gap repair upon homologous recombination at the endogenous Acadm locus resulting in a duplication of exons 8, 9, and 10 at the disrupted allele.\n(B) Southern blot analysis of EcoRI-digested genomic DNA from ES cells screened by PCR. Probe A, a DNA fragment consisting of a portion of exon 10 that is not present in the targeting vector, hybridizes to an endogenous 3.1-kb fragment and, upon homologous recombination, to a 13.2-kb fragment. Lane 1 represents a wild-type ES cell line, and Lane 2 and 3 represent targeted ES cell lines. ES cell clones were screened by PCR (data not shown) and confirmed by Southern blot analysis. Southern blot analysis used an exon 10 probe (probe A), not present in the targeting vector, hybridized to a 13.2-kb band in addition to the 3.1-kb endogenous band indicating targeted insertion of the vector at the Acadm locus (Figure 1B). Correctly targeted ES cell clones were microinjected into B6 (C57BL/6NTac) blastocysts to generate chimeric mice. Chimeric mice were backcrossed to both 129P2 and B6 inbred mice to produce MCAD+/− and eventually MCAD−/− mice on a B6/129 mixed background. The studies described here were conducted exclusively on the B6/129 mixed background compared with littermate controls or B6/129 control groups maintained by intercrosses as were the mutants. Perpetuating this mutation as a congenic mutant line on the 129P2 background proved impractical. The 129P2 mice were poor breeders as wild-types, and when introduced, the Acadm mutation was nearly lost on this background because of the high rate of neonatal death. Because of the molecular structure of the targeted allele, it proved virtually impossible to distinguish all three potential genotypes. We could clearly detect the presence or absence of the targeted allele, however, whether a particular mouse was MCAD−/− or MCAD+/− could not be determined by Southern blot or PCR of genomic DNA. Ultimately MCAD−/− mice were ascertained by immunoblot analysis of offspring with subsequent perpetuation of MCAD−/− and MCAD+/+ mice as separate groups."}