Zhao R, Fahs S A, Weiler H, Duncan S A
Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd, WI 53226, USA.
BMC Dev Biol. 2001;1:10. doi: 10.1186/1471-213x-1-10. Epub 2001 Jun 19.
Expression of transgenes in mice requires transcriptional regulatory elements that direct expression in a chosen cell type. Unfortunately, the availability of well-characterized promoters that direct bona-fide expression of transgenes in transgenic mice is limited. Here we described a method that allows highly efficient targeting of transgenes to a preselected locus in ES cells.
A pgk-LoxP-Neo cassette was introduced into a desired genomic locus by homologous recombination in ES cells. The pgk promoter was then removed from the targeted ES cells by Cre recombinase thereby restoring the ES cells' sensitivity to G418. We demonstrated that transgenes could be efficiently introduced into this genomic locus by reconstituting a functional Neo gene.
This approach is simple and extremely efficient in facilitating the introduction of single-copy transgenes into defined genomic loci. The availability of such an approach greatly enhances the ease of using endogenous regulatory elements to control transgene expression and, in turn, expands the repertoire of elements available for transgene expression.
在小鼠中表达转基因需要转录调控元件来指导在特定细胞类型中的表达。不幸的是，在转基因小鼠中指导转基因真正表达的特征明确的启动子的可用性有限。在此，我们描述了一种方法，该方法可将转基因高效靶向到胚胎干细胞（ES细胞）中的预选位点。
通过ES细胞中的同源重组，将一个磷酸甘油酸激酶（pgk）-LoxP-新霉素（Neo）盒引入到所需的基因组位点。然后通过Cre重组酶从靶向的ES细胞中去除pgk启动子，从而恢复ES细胞对G418的敏感性。我们证明，通过重建功能性Neo基因，转基因可以有效地引入到这个基因组位点。
这种方法在促进将单拷贝转基因引入特定基因组位点方面简单且极其有效。这种方法的可用性极大地提高了使用内源性调控元件来控制转基因表达的便利性，进而扩大了可用于转基因表达的元件库。
