State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Int J Biol Sci. 2010 Dec 2;6(7):756-68. doi: 10.7150/ijbs.6.756.
This study focused on concatemer formation and integration pattern of transgenes in zebrafish embryos. A reporter plasmid based on enhanced green fluorescent protein (eGFP) driven by Cytomegalovirus (CMV) promoter, pCMV-pax6in-eGFP, was constructed to reflect transgene behavior in the host environment. After removal of the insertion fragment by double digestion with various combinations of restriction enzymes, linearized pCMV-pax6in-eGFP vectors were generated with different combinations of 5'-protruding, 3'-protruding, and blunt ends that were microinjected into zebrafish embryos. Repair of double-strand breaks (DSBs) was monitored by GFP expression following religation of the reporter gene. One-hundred-and-ninety-seven DNA fragments were amplified from GFP-positive embryos and sequenced to analyze the repair characteristics of different DSB end combinations. DSBs involving blunt and asymmetric protruding ends were repaired efficiently by direct ligation of blunt ends, ligation after blunting and fill-in, or removed by cutting. Repair of DSBs with symmetric 3'-3' protrusions was less efficient and utilized template-directed repair. The results suggest that non-homologous end joining (NHEJ) was the principal mechanism of exogenous gene concatemer formation and integration of transgenes into the genome of transgenic zebrafish.
本研究专注于斑马鱼胚胎中外源基因的串联体形成和整合模式。构建了一种基于增强型绿色荧光蛋白（eGFP）的报告质粒，由巨细胞病毒（CMV）启动子驱动，pCMV-pax6in-eGFP，用于反映宿主环境中外源基因的行为。通过用各种组合的限制酶进行双酶切去除插入片段后，生成具有不同 5'突出、3'突出和钝端组合的线性化 pCMV-pax6in-eGFP 载体，这些载体被微注射到斑马鱼胚胎中。通过 GFP 表达监测报告基因的重连接来监测双链断裂（DSB）的修复。从 GFP 阳性胚胎中扩增了 197 个 DNA 片段并进行测序，以分析不同 DSB 末端组合的修复特征。涉及钝端和不对称突出端的 DSB 可以通过钝端的直接连接、钝化后连接和填充修复或切割来有效修复。具有对称 3'-3'突出的 DSB 的修复效率较低，并且利用模板指导修复。结果表明，非同源末端连接（NHEJ）是外源基因串联体形成和转基因整合到转基因斑马鱼基因组中的主要机制。
