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AlphaA-crystallin R49Cneo mutation influences the architecture of lens fiber cell membranes and causes posterior and nuclear cataracts in mice.

Andley UP - BMC Ophthalmol (2009)

Bottom Line: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency.By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed.It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, Missouri, USA. andley@vision.wustl.edu

ABSTRACT

Background: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency. The R49C mutation in the alphaA-crystallin protein is linked with non-syndromic, hereditary human cataracts in a four-generation Caucasian family.

Methods: This study describes a mouse cataract model generated by insertion of a neomycin-resistant (neor) gene into an intron of the gene encoding mutant R49C alphaA-crystallin. Mice carrying the neor gene and wild-type Cryaa were also generated as controls. Heterozygous knock-in mice containing one wild type gene and one mutated gene for alphaA-crystallin (WT/R49Cneo) and homozygous knock-in mice containing two mutated genes (R49Cneo/R49Cneo) were compared.

Results: By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed. WT/R49Cneo mice demonstrated severe posterior cataracts at 9 months of age, with considerable posterior nuclear migration evident in histological sections. R49Cneo/R49Cneo mice demonstrated nearly complete lens opacities by 5 months of age. In contrast, R49C mice in which the neor gene was deleted by breeding with CreEIIa mice developed lens abnormalities at birth, suggesting that the neor gene may suppress expression of mutant R49C alphaA-crystallin protein.

Conclusion: It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

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Plasmid construct used to generate R49Cneo gene knock-in mice. The 5' and 3' arms of the αA-crystallin gene (Cryaa) were cloned into a vector containing the floxed neomycin (neo) cDNA. Mutagenesis was performed to mutate amino acid arginine 49 of αA-crystallin to cysteine (R49C). The asterisk above exon 1 indicates the mutation. The numbered blue rectangles indicate exons. The filled triangles are loxP sites and X denotes the XhoI site. Mouse embryonic stem (ES) cells SCC-10 were electroporated with the mutant plasmid, and clones testing positive for neo were identified and used to generate R49Cneo αA-crystallin knock-in mice (WT/R49Cneo). One clone containing wild type (WT) αA-crystallin cDNA and neo was also analyzed and used to generate mice with the neo allele but no mutation (WT/WTneo).
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Figure 1: Plasmid construct used to generate R49Cneo gene knock-in mice. The 5' and 3' arms of the αA-crystallin gene (Cryaa) were cloned into a vector containing the floxed neomycin (neo) cDNA. Mutagenesis was performed to mutate amino acid arginine 49 of αA-crystallin to cysteine (R49C). The asterisk above exon 1 indicates the mutation. The numbered blue rectangles indicate exons. The filled triangles are loxP sites and X denotes the XhoI site. Mouse embryonic stem (ES) cells SCC-10 were electroporated with the mutant plasmid, and clones testing positive for neo were identified and used to generate R49Cneo αA-crystallin knock-in mice (WT/R49Cneo). One clone containing wild type (WT) αA-crystallin cDNA and neo was also analyzed and used to generate mice with the neo allele but no mutation (WT/WTneo).

Mentions: While constructing a gene knock-in mouse model of R49C αA-crystallin, we generated mice carrying a floxed neor gene in an intron of the Cryaa gene (Figure 1). Mating these to a mouse expressing Cre recombinase resulted in removal of the neo cassette, leaving the loxP site adjacent to exon 1 of the Cryaa gene, and producing a functional but less active mutant allele [42,55]. Two lines of R49Cneo mice, R49CneoKI 3 and R49CneoKI 4, were generated from a single ES cell clone, and the results obtained from these two lines were very similar. The knock-in mice were viable and bred normally. Heterozygous WT/R49Cneo mice were intercrossed to produce R49Cneo/R49Cneo homozygous offspring. A control mouse line was also generated from an ES cell clone that had the neomycin cassette inserted, but lacked the R49C αA-crystallin mutation. These mice did not have the altered lens phenotype, and served as a useful control. We speculated based on previous work by others [58-60] that the neor gene might exhibit diminished activity of mutant R49C gene that would provide novel insights into the biology of R49C αA-crystallin in vivo. To determine the effect of the R49Cneo gene on α-crystallin protein expression, lenses were analyzed by gel permeation chromatography. Figure 2 shows the chromatography profile of water-soluble lens proteins of WT/WTneo and WT/R49Cneo mice measured by absorbance measurements at 280 nm. The analysis showed a 30% decrease in expression of total α-crystallin protein in WT/R49Cneo heterozygous lens as compared with wild type lenses. The expression of β-crystallins also decreased whereas γ-crystallin expression was not appreciably affected. As compared with WT/R49C lenses [42], α-crystallin decreased more in the WT/R49Cneo lenses.


AlphaA-crystallin R49Cneo mutation influences the architecture of lens fiber cell membranes and causes posterior and nuclear cataracts in mice.

Andley UP - BMC Ophthalmol (2009)

Plasmid construct used to generate R49Cneo gene knock-in mice. The 5' and 3' arms of the αA-crystallin gene (Cryaa) were cloned into a vector containing the floxed neomycin (neo) cDNA. Mutagenesis was performed to mutate amino acid arginine 49 of αA-crystallin to cysteine (R49C). The asterisk above exon 1 indicates the mutation. The numbered blue rectangles indicate exons. The filled triangles are loxP sites and X denotes the XhoI site. Mouse embryonic stem (ES) cells SCC-10 were electroporated with the mutant plasmid, and clones testing positive for neo were identified and used to generate R49Cneo αA-crystallin knock-in mice (WT/R49Cneo). One clone containing wild type (WT) αA-crystallin cDNA and neo was also analyzed and used to generate mice with the neo allele but no mutation (WT/WTneo).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724435&req=5

Figure 1: Plasmid construct used to generate R49Cneo gene knock-in mice. The 5' and 3' arms of the αA-crystallin gene (Cryaa) were cloned into a vector containing the floxed neomycin (neo) cDNA. Mutagenesis was performed to mutate amino acid arginine 49 of αA-crystallin to cysteine (R49C). The asterisk above exon 1 indicates the mutation. The numbered blue rectangles indicate exons. The filled triangles are loxP sites and X denotes the XhoI site. Mouse embryonic stem (ES) cells SCC-10 were electroporated with the mutant plasmid, and clones testing positive for neo were identified and used to generate R49Cneo αA-crystallin knock-in mice (WT/R49Cneo). One clone containing wild type (WT) αA-crystallin cDNA and neo was also analyzed and used to generate mice with the neo allele but no mutation (WT/WTneo).
Mentions: While constructing a gene knock-in mouse model of R49C αA-crystallin, we generated mice carrying a floxed neor gene in an intron of the Cryaa gene (Figure 1). Mating these to a mouse expressing Cre recombinase resulted in removal of the neo cassette, leaving the loxP site adjacent to exon 1 of the Cryaa gene, and producing a functional but less active mutant allele [42,55]. Two lines of R49Cneo mice, R49CneoKI 3 and R49CneoKI 4, were generated from a single ES cell clone, and the results obtained from these two lines were very similar. The knock-in mice were viable and bred normally. Heterozygous WT/R49Cneo mice were intercrossed to produce R49Cneo/R49Cneo homozygous offspring. A control mouse line was also generated from an ES cell clone that had the neomycin cassette inserted, but lacked the R49C αA-crystallin mutation. These mice did not have the altered lens phenotype, and served as a useful control. We speculated based on previous work by others [58-60] that the neor gene might exhibit diminished activity of mutant R49C gene that would provide novel insights into the biology of R49C αA-crystallin in vivo. To determine the effect of the R49Cneo gene on α-crystallin protein expression, lenses were analyzed by gel permeation chromatography. Figure 2 shows the chromatography profile of water-soluble lens proteins of WT/WTneo and WT/R49Cneo mice measured by absorbance measurements at 280 nm. The analysis showed a 30% decrease in expression of total α-crystallin protein in WT/R49Cneo heterozygous lens as compared with wild type lenses. The expression of β-crystallins also decreased whereas γ-crystallin expression was not appreciably affected. As compared with WT/R49C lenses [42], α-crystallin decreased more in the WT/R49Cneo lenses.

Bottom Line: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency.By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed.It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St Louis, Missouri, USA. andley@vision.wustl.edu

ABSTRACT

Background: AlphaA-crystallin (CRYAA/HSPB4), a major component of all vertebrate eye lenses, is a small heat shock protein responsible for maintaining lens transparency. The R49C mutation in the alphaA-crystallin protein is linked with non-syndromic, hereditary human cataracts in a four-generation Caucasian family.

Methods: This study describes a mouse cataract model generated by insertion of a neomycin-resistant (neor) gene into an intron of the gene encoding mutant R49C alphaA-crystallin. Mice carrying the neor gene and wild-type Cryaa were also generated as controls. Heterozygous knock-in mice containing one wild type gene and one mutated gene for alphaA-crystallin (WT/R49Cneo) and homozygous knock-in mice containing two mutated genes (R49Cneo/R49Cneo) were compared.

Results: By 3 weeks, WT/R49Cneo mice exhibited large vacuoles in the cortical region 100 mum from the lens surface, and by 3 months posterior and nuclear cataracts had developed. WT/R49Cneo mice demonstrated severe posterior cataracts at 9 months of age, with considerable posterior nuclear migration evident in histological sections. R49Cneo/R49Cneo mice demonstrated nearly complete lens opacities by 5 months of age. In contrast, R49C mice in which the neor gene was deleted by breeding with CreEIIa mice developed lens abnormalities at birth, suggesting that the neor gene may suppress expression of mutant R49C alphaA-crystallin protein.

Conclusion: It is apparent that modification of membrane and cell-cell interactions occurs in the presence of the alphaA-crystallin mutation and rapidly leads to lens cell pathology in vivo.

Show MeSH
Related in: MedlinePlus