Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

被引：24

作者：

Misra R.P. ^{[2
]}

Bronson S.K. ^{[3
]}

Xiao Q. ^{[2
]}

Garrison W. ^{[1
]}

Li J. ^{[1
]}

Zhao R. ^{[1
]}

Duncan S.A. ^{[1
]}

机构：

[1] Dept. Cell. Biol. Neurobiol./Anat., Medical College of Wisconsin, Milwaukee, WI 53226

[2] Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226

[3] Dept. of Cellular/Molec. Physiology, The Penn State Coll. of Med. HI 66, The Milton S. Hershey Medical Center, Hershey, PA 17033-0850

来源：

BMC Biotechnology | / 1卷 / 1期

关键词：

Hprt Gene; Transcriptional Regulatory Element; Hprt Locus; Tetraploid Embryo; Restriction Fragment Polymorphism;

D O I：

10.1186/1472-6750-1-12

中图分类号：

学科分类号：

摘要：

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 α-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. © 2001 Misra et al; licensee BioMed Central Ltd.

引用

页数：9

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