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


CONCLUSIONS
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.
Zhao
R
R
Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, 8701 Watertown Plank Rd, WI 53226, USA. rzhao@mcw.edu
Fahs
S A
SA
Weiler
H
H
Duncan
S A
SA
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology; D018390:Gene Targeting / Q000379:methods; D005819:Genetic Markers / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D051541:Hepatocyte Nuclear Factor 3-alpha; D019426:Integrases / Q000235:genetics; D051379:Mice; D016254:Mutagenesis, Insertional / Q000379:methods*; D009355:Neomycin / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D013234:Stem Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors; D019076:Transgenes / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; C496500:Foxa1 protein, mouse; D005819:Genetic Markers; D051541:Hepatocyte Nuclear Factor 3-alpha; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D010735:Phosphoglycerate Kinase; C045073:Cre recombinase; D019426:Integrases; D009355:Neomycin