Uh Mitchell, Khattra Jaswinder, Devlin Robert H
Centre for Aquaculture and Environmental Research, Fisheries and Oceans Canada, West Vancouver, BC, Canada.
Transgenic Res. 2006 Dec;15(6):711-27. doi: 10.1007/s11248-006-9016-4. Epub 2006 Sep 2.
Currently, little information is available regarding the molecular organization of integrated transgenes in genetically-engineered fish. We performed a detailed structural analysis of an inserted transgene in one strain (M77) of transgenic coho salmon (Oncorhynchus kisutch) containing a salmon growth hormone gene construct (OnMTGH1). Microinjected DNA was found to have inserted into a single site in the coho salmon genome, and was organized with four complete internal copies and two partial terminal copies of the OnMTGH1 construct. All construct copies were organized in a direct-tandem (head-to-tail) repeat fashion in strain M77 and five additional strains (one also possessed a second recombinant junction fragment). For strain M77, the junctions between the transgene insert and the insertion point within the wild-type genome were cloned from strain-specific cosmid libraries and sequenced, revealing that the transgene insertion was accompanied by a deletion of 587 bp of wild-type DNA as well as a small insertion (19 bp) of unknown DNA upstream and a 14 bp direct- tandem duplication of sequence downstream. Upstream and downstream wild-type DNA sequence contained several repetitive sequence elements based on Southern blot analysis and homology to repetitive sequences in GenBank. In the downstream flank, a pseudogene sequence was also identified which has high homology to the CA membrane protein gene from Schistosoma japonicum, a parasite closely related to Sanguinicola sp. parasites which infect salmonids. Whether the presence of an inserted transgene and the presence of potentially horizontally-transmitted DNA are indicative of a genomic region with a predisposition for insertion of foreign DNA requires further study. The information derived from this transgene structure provides information useful for comparison to other transgenic organisms and for determination of the mechanism of transgene integration in lower vertebrates.
目前，关于基因工程鱼中整合转基因的分子组织的信息很少。我们对转基因银大麻哈鱼（Oncorhynchus kisutch）的一个品系（M77）中插入的转基因进行了详细的结构分析，该品系含有一个鲑鱼生长激素基因构建体（OnMTGH1）。发现显微注射的DNA插入到了银大麻哈鱼基因组的一个位点，并且由OnMTGH1构建体的四个完整内部拷贝和两个部分末端拷贝组成。在品系M77和另外五个品系中，所有构建体拷贝均以直接串联（头对尾）重复的方式排列（其中一个品系还拥有第二个重组连接片段）。对于品系M77，从品系特异性粘粒文库中克隆并测序了转基因插入片段与野生型基因组内插入点之间的连接点，结果表明转基因插入伴随着587 bp野生型DNA的缺失，以及上游未知DNA的小插入（19 bp）和下游14 bp的直接串联重复序列。根据Southern印迹分析和与GenBank中重复序列的同源性，上游和下游野生型DNA序列包含几个重复序列元件。在下游侧翼，还鉴定出一个假基因序列，它与日本血吸虫的CA膜蛋白基因具有高度同源性，日本血吸虫是一种与感染鲑科鱼类的血居吸虫属寄生虫密切相关的寄生虫。插入的转基因的存在以及潜在水平传播的DNA的存在是否表明基因组区域易于插入外源DNA，这需要进一步研究。从该转基因结构获得的信息为与其他转基因生物进行比较以及确定低等脊椎动物中转基因整合的机制提供了有用的信息。
