Emergency prevention of extinction of a transgenic allele in a less-fertile transgenic mouse line by crossing with an inbred or outbred mouse strain coupled with assisted reproductive technologies
Anna Mayer A , Diana Bulian A , Hagen Scherb B , Martin Hrabé de Angelis C , Jörg Schmidt A and Esther Mahabir A DA Department of Comparative Medicine, GSF–National Research Center for Environment and Health, 85764 Neuherberg, Germany.
B Institute of Biomathematics and Biometry, GSF–National Research Center for Environment and Health, 85764 Neuherberg, Germany.
C Institute of Experimental Genetics, GSF–National Research Center for Environment and Health, 85764 Neuherberg, Germany.
D Corresponding author. Email: mahabir@gsf.de
Reproduction, Fertility and Development 19(8) 984-994 https://doi.org/10.1071/RD06161
Submitted: 7 December 2006 Accepted: 11 September 2007 Published: 30 October 2007
Abstract
Certain transgenic mouse lines are difficult to breed or archive and, consequently, their transgenes become lost. We examined a C57BL/6 mouse line (B6-tg), transgenic for green fluorescent protein (GFP) with low fertility, and its crosses with the more prolific inbred C3HeB/FeJ (C3) and outbred Swiss (SW) strains in order to assess the possibility of emergency prevention of extinction of a transgenic allele by using assisted reproductive technologies (ART). Out-crossing was performed by natural mating or in vitro fertilisation (IVF) with heterozygous mice. Most of the crossing combinations resulted in improved archiving and rederivation efficiencies of the transgenic allele. Natural crossing increased both mean litter size by two to three pups and the superovulatory rate from 69% for B6-tg to 70–90% for females from the out-crosses. Each plug-positive B6-tg female yielded an average of 4.6 two-cell embryos, whereas females from the out-crosses produced three- to fivefold that amount. After thawing, 13% of B6-tg embryos and 6–12% of out-crossed embryos developed into transgenic pups after transfer into recipients. After IVF with cryopreserved spermatozoa, cleavage rates were 4% for B6-tg, 22–37% for B6-tg oocytes out-crossed with C3 and SW spermatozoa, 9–49% for gametes from out-crossed mice and 28–44% for back-crosses with B6 oocytes. Transgenic pups were not derived from IVF with B6-tg gametes when either fresh or cryopreserved spermatozoa were used. Rederivation efficiencies were 7% and 4% from out-crosses of B6-tg oocytes with C3 and SW spermatozoa, respectively, 6–22% for gametes from out-crossed mice and 4–10% for the back-crosses. Although out-crossing changes the original genetic background, the strategy of crossing coupled with ART prevents the extinction of an allele of interest, especially where archiving and rederivation of the transgenic line fail.
Additional keywords: cryopreservation, rederivation, transgenic allele rescue.
Acknowledgements
The authors thank Dr J. Favour for critically reviewing the manuscript, R. Schmöller and K. Markoullis for technical assistance and S. Scharold, M. Wöhl and I. Krüger for help in managing the mouse colonies. This work was supported by the National Genome Research Network (NGFN), Germany, and by the EU Commission.
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