研究目的
To recreate the human lamellar cataract phenotype in transgenic mice for investigation of this human pathology at a level not possible previously.
研究成果
Recreation of the human lamellar cataract phenotype in mice allows investigation of this human pathology at a level not possible previously and points to the relevance of fiber cell heterogeneity dictated by fiber cell–specific gene activity in the biogenesis of the lamellar cataract.
研究不足
The molecular pathogenesis, which cannot be observed with the slit-lamp, precedes the morphologic pathology. The opacities were observed by slit-lamp at 21 days while the molecular changes are seen at PND02, suggesting global changes in the lens fiber cells, yet the pathology matures to a cataractous, morphologically recognizable phenotype only in specific lamellae.
1：Experimental Design and Method Selection
Used bacterial artificial chromosome (BAC) transgenesis to express a hybrid gene: Hsf4 (DBD)–enhanced green fluorescent protein (EGFP), by recombineering EGFP sequences into the DBD of the Hsf4 gene, to interfere with the DNA binding properties of Hsf4.
2：Sample Selection and Data Sources
Four BAC DNAs of various lengths were used for the manipulation of the Hsf4 gene through recombineering. Transgenic mice were generated and maintained for the study.
3：List of Experimental Equipment and Materials
Bacterial Artificial Chromosome (BAC) clones, E. coli DH10B, pBSK-EGFP -FRT1-Tn5-neo-FRT2 plasmid, PCR primers, slit-lamp ophthalmoscope (BM900; Hagg-Streit), Cannon camera (EOS Rebel T2i; Canon), laser scanning confocal microscope (FluoView FV1000; Olympus Corporation).
4：Experimental Procedures and Operational Workflow
BAC recombineering, generation and maintenance of transgenic mice, slit-lamp ophthalmoscopy, genotyping and copy number determination, preparation of HSF4-EGFP reporter plasmids, cell culture and transfection, histology, immunofluorescence, and immunoblotting, 2D gel electrophoresis and quantitative RT-PCR (RT-qPCR), counting of epithelial cells and fiber cell nuclei.
5：Data Analysis Methods
Statistical analysis using Student’s t-tests to compare differences between the groups (WT versus transgenic lens sections). RNA was analyzed from two or three mice per line as indicated in figure legends.
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