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Growth and muscle defects in mice lacking adult myosin heavy chain genes.

Acakpo-Satchivi LJ, Edelmann W, Sartorius C, Lu BD, Wahr PA, Watkins SC, Metzger JM, Leinwand L, Kucherlapati R - J. Cell Biol. (1997)

Bottom Line: The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical.Both strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product.Most striking is that while both strains exhibit physiological defects in isolated muscles, the defects are distinct.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Albert Einstein College of Medicine, New York 10461, USA.

ABSTRACT
The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb mutants are generally milder than in the MyHC-IId/x strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for expression of the two genes. Most striking is that while both strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb mice has significantly reduced ability to generate force while IId mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.

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The MyHC-IIb and IId/x targeting strategies and Southern blot analysis of F2 mice. Targeting  vector, wild-type and recombinant loci are shown  with relevant restriction sites. (A) A 6-kb HindIII  fragment containing the first five exons of the MyHC-IIb gene was subcloned from the λ10a clone. The  PGK-neomycin cassette was inserted into the third  exon, downstream of the translational start site, and  in the opposite orientation to the MyHC gene. The  PGK-thymidine kinase gene for negative selection  was placed 3′ of the construct as shown. Homologously recombined clones were assayed for by EcoRI  Southern blot analysis using a probe 3′ of the construct,  as indicated. (B) A 11-kb EcoRI fragment containing  the first five exons of the MyHC-IId/x gene was subcloned from the λ19a clone. The PGK-neomycin cassette was inserted into a unique BspEI site, downstream of the translational start site, in the same  orientation as the MyHC gene. Homologous recombination was assayed by BglII Southern Blot analysis  using a probe 5′ of the targeting construct, as shown.  (C) The first panel depicts a EcoRI southern blot  analysis of F2 mice from the IIb targeting experiment.  Wild-type, heterozygous and homozygous  animals are shown. The second panel depicts a BglII  southern blot analysis of F2 mice from the IId/x targeting experiment.
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Figure 1: The MyHC-IIb and IId/x targeting strategies and Southern blot analysis of F2 mice. Targeting vector, wild-type and recombinant loci are shown with relevant restriction sites. (A) A 6-kb HindIII fragment containing the first five exons of the MyHC-IIb gene was subcloned from the λ10a clone. The PGK-neomycin cassette was inserted into the third exon, downstream of the translational start site, and in the opposite orientation to the MyHC gene. The PGK-thymidine kinase gene for negative selection was placed 3′ of the construct as shown. Homologously recombined clones were assayed for by EcoRI Southern blot analysis using a probe 3′ of the construct, as indicated. (B) A 11-kb EcoRI fragment containing the first five exons of the MyHC-IId/x gene was subcloned from the λ19a clone. The PGK-neomycin cassette was inserted into a unique BspEI site, downstream of the translational start site, in the same orientation as the MyHC gene. Homologous recombination was assayed by BglII Southern Blot analysis using a probe 5′ of the targeting construct, as shown. (C) The first panel depicts a EcoRI southern blot analysis of F2 mice from the IIb targeting experiment. Wild-type, heterozygous and homozygous animals are shown. The second panel depicts a BglII southern blot analysis of F2 mice from the IId/x targeting experiment.

Mentions: The MyHC-IIb targeting vector (Fig. 1 A) was constructed from a genomic fragment which contained part of the 5′ end of the MyHC-IIb gene, including the first two non-coding (exons 1 and 2), and the first three coding exons (exons 3–5). A 1.8-kb fragment containing the PGK-Neo cassette was inserted into a BspEI site in the third exon of the MyHC-IIb gene. The transcriptional orientation of the Neo cassette was opposite to that of the MyHC gene. A PGK-TK cassette was inserted downstream of the modified gene for negative selection.


Growth and muscle defects in mice lacking adult myosin heavy chain genes.

Acakpo-Satchivi LJ, Edelmann W, Sartorius C, Lu BD, Wahr PA, Watkins SC, Metzger JM, Leinwand L, Kucherlapati R - J. Cell Biol. (1997)

The MyHC-IIb and IId/x targeting strategies and Southern blot analysis of F2 mice. Targeting  vector, wild-type and recombinant loci are shown  with relevant restriction sites. (A) A 6-kb HindIII  fragment containing the first five exons of the MyHC-IIb gene was subcloned from the λ10a clone. The  PGK-neomycin cassette was inserted into the third  exon, downstream of the translational start site, and  in the opposite orientation to the MyHC gene. The  PGK-thymidine kinase gene for negative selection  was placed 3′ of the construct as shown. Homologously recombined clones were assayed for by EcoRI  Southern blot analysis using a probe 3′ of the construct,  as indicated. (B) A 11-kb EcoRI fragment containing  the first five exons of the MyHC-IId/x gene was subcloned from the λ19a clone. The PGK-neomycin cassette was inserted into a unique BspEI site, downstream of the translational start site, in the same  orientation as the MyHC gene. Homologous recombination was assayed by BglII Southern Blot analysis  using a probe 5′ of the targeting construct, as shown.  (C) The first panel depicts a EcoRI southern blot  analysis of F2 mice from the IIb targeting experiment.  Wild-type, heterozygous and homozygous  animals are shown. The second panel depicts a BglII  southern blot analysis of F2 mice from the IId/x targeting experiment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140209&req=5

Figure 1: The MyHC-IIb and IId/x targeting strategies and Southern blot analysis of F2 mice. Targeting vector, wild-type and recombinant loci are shown with relevant restriction sites. (A) A 6-kb HindIII fragment containing the first five exons of the MyHC-IIb gene was subcloned from the λ10a clone. The PGK-neomycin cassette was inserted into the third exon, downstream of the translational start site, and in the opposite orientation to the MyHC gene. The PGK-thymidine kinase gene for negative selection was placed 3′ of the construct as shown. Homologously recombined clones were assayed for by EcoRI Southern blot analysis using a probe 3′ of the construct, as indicated. (B) A 11-kb EcoRI fragment containing the first five exons of the MyHC-IId/x gene was subcloned from the λ19a clone. The PGK-neomycin cassette was inserted into a unique BspEI site, downstream of the translational start site, in the same orientation as the MyHC gene. Homologous recombination was assayed by BglII Southern Blot analysis using a probe 5′ of the targeting construct, as shown. (C) The first panel depicts a EcoRI southern blot analysis of F2 mice from the IIb targeting experiment. Wild-type, heterozygous and homozygous animals are shown. The second panel depicts a BglII southern blot analysis of F2 mice from the IId/x targeting experiment.
Mentions: The MyHC-IIb targeting vector (Fig. 1 A) was constructed from a genomic fragment which contained part of the 5′ end of the MyHC-IIb gene, including the first two non-coding (exons 1 and 2), and the first three coding exons (exons 3–5). A 1.8-kb fragment containing the PGK-Neo cassette was inserted into a BspEI site in the third exon of the MyHC-IIb gene. The transcriptional orientation of the Neo cassette was opposite to that of the MyHC gene. A PGK-TK cassette was inserted downstream of the modified gene for negative selection.

Bottom Line: The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical.Both strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product.Most striking is that while both strains exhibit physiological defects in isolated muscles, the defects are distinct.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Albert Einstein College of Medicine, New York 10461, USA.

ABSTRACT
The three adult fast myosin heavy chains (MyHCs) constitute the vast majority of the myosin in adult skeletal musculature, and are >92% identical. We describe mice carrying mutations in each of two predominant adult fast MyHC genes, IIb and IId/x. Both strains exhibit growth and muscle defects, but the defects are different between the two strains and do not correlate with the abundance or distribution of each gene product. For example, despite the fact that MyHC-IIb accounts for >70% of the myosin in skeletal muscle and shows the broadest distribution of expression, the phenotypes of IIb mutants are generally milder than in the MyHC-IId/x strain. In addition, in a muscle which expresses both IIb and IId/x MyHC in wild-type mice, the histological defects are completely different for expression of the two genes. Most striking is that while both strains exhibit physiological defects in isolated muscles, the defects are distinct. Muscle from IIb mice has significantly reduced ability to generate force while IId mouse muscle generates normal amounts of force, but has altered kinetic properties. Many of the phenotypes demonstrated by these mice are typical in human muscle disease and should provide insight into their etiology.

Show MeSH
Related in: MedlinePlus