The Laboratory of Ophthalmology and Vision Science, Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zheng Zhou, China.
Department of Medical Genetics and Cell Biology, School of Basic Medical Sciences, Zhengzhou University, Zheng Zhou, China.
Mol Vis. 2024 Mar 19;30:123-136. eCollection 2024.
Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens.
The pTol2 :Cre-polyA-:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish.
In this study, we generated a transgenic zebrafish line, zTg(:Cre-:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific promoter. zTg(:Cre-:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(:Cre-:EGFP) embryos were injected with the -flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(:Cre-:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses.
We established a zTg(:Cre-:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.
斑马鱼是研究晶状体发育和先天性白内障的常用模型。然而，由于缺乏晶状体-Cre-转基因斑马鱼，无法使用 Cre/LoxP 系统在斑马鱼晶状体中特异性缺失基因。本研究旨在构建一种在晶状体中特异性表达 Cre 重组酶的转基因斑马鱼系。
构建 pTol2:Cre-polyA-:EGFP（增强型绿色荧光蛋白）质粒，并将其与 Tol2 转座酶共注射到一至二细胞期野生型（WT）斑马鱼胚胎中。采用全胚胎原位杂交（ISH）、组织切片、苏木精和伊红染色、Western blot、裂隙灯观察和网格传输试验，分析转基因斑马鱼的 Cre 表达、晶状体结构和晶状体透明度。
本研究构建了一种转基因斑马鱼系 zTg(:Cre-:EGFP)，其中 Cre 重组酶和 EGFP 由晶状体特异性启动子驱动。zTg(:Cre-:EGFP)在 22 hpf 时开始在晶状体中特异性表达 Cre 和 EGFP，当 zTg(:Cre-:EGFP)胚胎被注射 -flanked RFP 质粒时，该异位 Cre 可以有效地、特异性地从晶状体中删除 RFP 信号。Cre 和 EGFP 的过表达不会损害斑马鱼的发育或晶状体透明度。因此，这种 zTg(:Cre-:EGFP)斑马鱼系是用于基因编辑的有用工具，特别是用于斑马鱼晶状体。
我们建立了一种 zTg(:Cre-:EGFP)斑马鱼系，可在晶状体组织中特异性表达活性 Cre 重组酶。这种转基因斑马鱼系可作为一种工具，用于在斑马鱼晶状体中特异性操纵基因。
