Site-specific recombination using an epitope tagged bacteriophage P1 Cre recombinase.
Site-specific recombination using an epitope tagged bacteriophage P1 Cre recombinase.
复制标题
使用表位标记的噬菌体 P1 Cre 重组酶进行位点特异性重组。
DOI：
10.1016/s0378-1119(98)00249-2
复制
发表时间：
1998
期刊：
影响因子：
3.5
通讯作者：
Kohan,DE
中科院分区：
文献类型：
--
作者：
Stricklett,PK;Nelson,RD;Kohan,DE
自从对噬菌体 P1 中 Cre 介导的位点特异性重组的最初描述（Sternberg, N., Hamilton, D., 1981 J. Mol. Biol., 150, 467-487）以来，Cre-lox 重组系统已被广泛用于操纵原核和真核基因组。不幸的是，很少有方法可以测量体内 Cre 蛋白的表达。我们构建了表达载体，其中Cre蛋白在羧基末端标记有单纯疱疹病毒(HSV)糖蛋白D外壳蛋白的11个氨基酸表位(Isola, V.J.、Eisenberg, R.J.、Siebert, G.R.、Heilman, C.J.、Wilcox, W.C.、Cohan, G.H., 1989. J. Virol. 63、2325-2334）。表位标签有助于使用市售抗体进行免疫荧光标记来检测体外和体内的 Cre 表达。表位标签不会干扰 Cre 重组酶活性或改变 loxP 位点之间的重组效率。我们已经在小鼠中证明，表达我们标记的 Cre 的转基因能够切除另一只转基因小鼠贡献的 loxP 侧翼序列。总之，我们开发了一种带有表位标记的 Cre 重组酶，该酶具有完全活性且易于检测。
Since the original description of Cre mediated site-specific recombination in bacteriophage P1 (Sternberg, N., Hamilton, D., 1981 J. Mol. Biol., 150, 467-487), the Cre-lox system of recombination has been widely used to manipulate prokaryotic and eukaryotic genomes. Unfortunately, there are few means available to measure Cre protein expression in vivo. We have constructed an expression vector wherein the Cre protein is tagged at the carboxy terminus with an 11-amino-acid epitope to the herpes simplex virus (HSV) glycoprotein D coat protein (Isola, V.J., Eisenberg, R.J., Siebert, G.R., Heilman, C.J., Wilcox, W.C., Cohan, G.H., 1989. J. Virol. 63, 2325-2334). The epitope tag facilitates detection of Cre expression in vitro and in vivo using immunofluorescent labeling with a commercially available antibody. The epitope tag does not interfere with Cre recombinase activity or alter recombination efficiency between loxP sites. We have shown in mice that a transgene expressing our tagged Cre is capable of excising a loxP flanked sequence contributed by another transgenic mouse. In summary, we have developed an epitope-tagged Cre recombinase that is fully active and readily detectable.