Nucleic Acids Research Advance Access originally published online on March 11, 2009
Nucleic Acids Research 2009 37(7):e55; doi:10.1093/nar/gkp112
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Nucleic Acids Research, 2009, Vol. 37, No. 7 e55
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells
1Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, 2Department of Biomedical Molecular Biology, Ghent University, 3Laboratory for Molecular Cancer Biology, VIB-UGent, 4Molecular and Cellular Oncology Unit, Department for Molecular Biomedical Research, 5Department of Plant Systems Biology, VIB, 6Department of Molecular Genetics, Ghent University, Gent, Belgium, 7Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and 8Department of Molecular and Medical Genetics, University of Toronto, Toronto, ON, Canada
*To whom correspondence should be addressed. Tel: +32 (0) 9 33 13 730; Fax: +32 (0) 9 33 13 609; Email: jody.haigh{at}dmbr.ugent.be
Received September 11, 2008. Revised January 15, 2009. Accepted February 10, 2009.
The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3–4 weeks by using Gateway® cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.
The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.