Methodology article

An efficient method to successively introduce transgenes into a given genomic locus in the mouse

Roong Zhao¹ , Scott A Fahs² , Hartmut Weiler² and Stephen A Duncan¹

¹Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd, WI 53226, USA

²Blood Research Institute, The Blood Center of Southeastern Wisconsin, 8272 Watertown Plank Rd., Milwaukee, Wisconsin 53226

author email corresponding author email

BMC Developmental Biology 2001, 1:10doi:10.1186/1471-213X-1-10

Published:	19 June 2001

Abstract

Background

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.

Results

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.

Conclusion

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.