Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation. BMC Biotechnol 2001;1:12
Date
01/10/2002Pubmed ID
11782291Pubmed Central ID
PMC64498DOI
10.1186/1472-6750-1-12Scopus ID
2-s2.0-3042609697 (requires institutional sign-in at Scopus site) 29 CitationsAbstract
BACKGROUND: Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites.
RESULTS: Here we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the alpha-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern.
CONCLUSION: Our results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This facilitates the comparative analysis of lethal alleles and thereby advances our ability to analyze gene function in mammals.
Author List
Misra RP, Bronson SK, Xiao Q, Garrison W, Li J, Zhao R, Duncan SAAuthor
Ravindra P. Misra PhD Associate Provost, Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AllelesAnimals
Cell Line
Embryo, Mammalian
Gene Deletion
Gene Dosage
Genes, Lethal
Genetic Complementation Test
Hypoxanthine Phosphoribosyltransferase
Mice
Mice, Transgenic
Mutagenesis, Site-Directed
Myocardium
Organ Specificity
Polyploidy
Promoter Regions, Genetic
Recombination, Genetic
Selection, Genetic
Stem Cells
Transgenes
Ventricular Myosins
