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Nan Zhou, Yanhua Qi; MIP mutation study in zebrafish by using Cas9 system. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5597.
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© ARVO (1962-2015); The Authors (2016-present)
This study aimed to identify genetic defects associated with congenital progressive punctate cataracts in a Chinese family. We established zebrafish model of the mutation to further explore the potential pathogenesis.
Linkage analysis was performed by microsatellite markers and candidate gene was screened by direct sequencing. The mutation was studied in knock out zebrafish established using Cas9/gRNA. RT-PCR and whole-mount in situ hybridization were performed on wild type and mutant zebrafish embryos at various developmental stages to determine the expression pattern. Paraffin section and HE staining was used to observe the changes in organization structure of lens in the knockout zebrafish.
Positive two-point LOD scores were obtained at marker D12S1622 (Zmax = 2.71 at θ = 0.0) and D12S90 (Zmax = 2.71 at θ = 0.0), which flank the MIP (Aqp0) gene on chromosomal region 12q13. Direct sequencing of MIP gene revealed a novel mutation (G>D) in exon 4 at nucleotide 644, which caused a substitution of glycine to aspartic acid at codon 215 (p.G215D). The wild type and mutant MIP were transfected with green fluorescent protein (GFP) into Hela cells separately, and it was found that the G215D mutant was aberrantly located in the cytoplasm instead of in the plasma membrane. In zebrafish, the mutant Aqp0b gene identified a -7bp deletion in exon 4. This deletion produces a frame-shift that results in the synthesis of a truncated protein in the six trans-membrane and the COOH-terminus of Aqp0b. The Aqp0b -/- zebrafish shows pericardial edema, small eye and abnormal lens without opacity, while Aqp0b /+ and wild type displays normal. RT-PCR and whole-mount in situ hybridization demonstrated no significant difference of Aqp0b mRNA expression in lens between Aqp0b -/- homozygotes and wild-type. Paraffin section and HE staining showed that Aqp0b -/- homozygotes developed small eyes and small, irregular round lens.
Our study presented a new MIP mutation that causes autosomal dominant congenital cataracts. This mutation doesn't result in changes in the expression of MIP, but result in the mutant protein aberrantly located in the cytoplasm instead of in the plasma membrane, which may lead to functional inactivation. Aqp0b -/- zebrafish mutant suggests that the loss function of Aqp0b is possibly complemented by its copy gene-Aqp0a, but it still can show the important role in the lens development and maintenance.
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