Ivics Z, Izsvák Z, Hackett P B
Department of Genetics & Cell Biology, University of Minnesota, St. Paul 55108.
Mol Mar Biol Biotechnol. 1993 Jun;2(3):162-73.
Manufacture of lines of fish containing specific transgenes is difficult because most fish that hatch from embryos injected with foreign DNA are mosaic; few have the transgenic DNA integrated in germ-line cells. To determine whether the process of integration of exogenously supplied DNA into fish genomes could be accelerated, we examined the ability of the Moloney murine leukemia virus (MoMLV) integration protein (IN) to function in embryonic zebrafish cells. We used partially purified IN from a baculovirus/insect cell expression system and unpurified IN from extracts of psi-2 mouse cells that carry a MoMLV provirus. Both forms of IN were able to enhance expression in zebrafish 10 days after fertilization. At day 14 of development, fish injected with IN had higher levels of transgenic DNA than control fish. The ability of IN to enhance integration of transgenic constructs was demonstrated by a ligation-mediated polymerase chain reaction procedure, which was employed to detect junction fragments of foreign and host genomic DNA, generated by IN-mediated integration.
制造含有特定转基因的鱼系很困难，因为大多数从注射了外源DNA的胚胎中孵化出的鱼都是嵌合体；很少有转基因DNA整合到生殖系细胞中。为了确定外源提供的DNA整合到鱼类基因组中的过程是否可以加速，我们研究了莫洛尼鼠白血病病毒（MoMLV）整合蛋白（IN）在斑马鱼胚胎细胞中的功能。我们使用了来自杆状病毒/昆虫细胞表达系统的部分纯化的IN和来自携带MoMLV前病毒的psi-2小鼠细胞提取物中的未纯化的IN。两种形式的IN都能够在受精后10天增强斑马鱼中的表达。在发育的第14天，注射了IN的鱼的转基因DNA水平高于对照鱼。通过连接介导的聚合酶链反应程序证明了IN增强转基因构建体整合的能力，该程序用于检测由IN介导的整合产生的外源和宿主基因组DNA的连接片段。
