Experiment
Mutations in the gene for destrin (Dstn), an actin depolymerizing factor, leads to corneal abnormalities in mice. The current study demonstrates that a quantitative trait locus (QTL) is responsible for genetic modification of corneal neovascularixation in Dstncorn1 mice.
A.BY-H2bc H2-T18f/SnJ-Dstncorn1/J (A.BY-Dstncorn1) mice were crossed with C57BL/6J wild type (B6) mice to generate (A.BY-Dstncorn1 x C57BL/6J)F1 mice. Progeny were selected for mice carrying the Dstncorn1 allele and backcrossed to B6 mice for 8 generations, and the heterozygous B6.Cg-Dstncorn1 mice were intercrossed. Homozygous mutant mice on the A.BY and C57BL/6J backgrounds were intercrossed and N2 progeny, (A.BY-Dstncorn1 x B6.Cg-Dstbcorn1)F1 x B6.Cg-Dstbcorn1, were used for analysis.
Immunohistochemistry was performed on whole cornea. Digital images of corneal flat mounts were collected using the Spot Image Analysis system. Vascularized area and total corneal area were measured using ImageJ software. The total corneal area was outlined, and the area of CD31-positive vessels within the cornea was then calculated and normalized to the total corneal area (expressed as a percentage of vascularized cornea) using thresholding analysis.
Initially a whole genome scan was performed using 66 N2 Dstncorn1 mice. Sixty-five markers throughout the genome were selected to distinguish between A.BY and B6 alleles. The R/qtl package was used to perform statistical analysis. LOD scores were calculated using the multiple imputation method with 1-cM steps, 1000 joint genotype distribution imputations and an assumed genotyping error rate of 0.01. After determining the presence of a possible QTL on Chr 3, an additional 135 N2 mice were genotyped with additional Chr 3 markers.
In the analysis of the phenotypic and genotypic data from the 66 N2 mice, aged 58 days, a major QTL region was identified on Chr 3. An additional 135 N2 mice were genotyped with additional Chr 3 markers confirming the existence of a significant QTL associated with modification of corneal neovascularization in Dstncorn1 mice. QTL Modn1 (modifier of Dstncorn1 neovascularization 1) peaked at marker D3Mit315, LOD=3.51, p=0.0071, with a 3 LOD support interval mapping from 8.0cM to 75.67cM. The A.BY allele at this locus had a significant effect in increasing the amount of neovascularization in Dstncorn1 mice in a semi-dominant inheritance pattern.
The validity of Modn1 was tested by crossing C57BL/6J-Chr3A/J/NaJ consomic mice with B6.Cg-Dstncorn1 mice generating F2 and F3 Dstncorn1 mutant mice with 3 different genotypes at the potential QTL locus on Chr 3. Significant increase in vascularization in mice carring one or two A/J alleles at Modn1 was observed.
Candidate genes and genes of interest are listed in Table 1, page 16 including the following Chromosome 3 genes known to influence actin dynamics: Arhgef11 and Ank2.
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