Abstract
Chromosomal deletions, as a genetic tool for functional genomics, remain underexploited for vertebrate stem cells mostly because presently available methods are too labor-intensive. To address this, we developed and validated a set of complementary retroviruses that creates a wide range of nested chromosomal deletions. When applied to mouse embryonic stem cells (ESCs), this retrovirus-based method yielded deletions ranging from 6 kb to 23 Mb (average 2.9 Mb), with an efficiency of 64% for drug-selected clones. Notably, several of the engineered ESC clones, mostly those with large deletions, showed major alteration in cell fate. In comparison to other methods that have also exploited retroviruses for chromosomal engineering, this modified strategy is more efficient and versatile because it bypasses the need for homologous recombination, and thus can be exploited for rapid and extensive functional screens in embryonic and adult stem cells.
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Acknowledgements
We thank P. Chartrand, M. Therrien and colleagues for critically reading the manuscript; V. Paradis, S. Harton and E. Milot from the transgenic facility of IRIC; J. Cowell, M. Rossi and D. McQuaid for the aCGH service (Roswell Park Cancer Institute); J.-P. Laverdure from the bioinformatic service of IRIC;C. Charbonneau from the imaging service of IRIC; N. Fradet, M. Fréchette, A. Fredette, E. St-Hilaire, T. MacRae, C. Rondeau and P. Lussier for technical assistance; A. Nagy for providing the R1 ESCs and the pCX-EYFP construct (Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto); A. Bradley for the pOG231 construct (Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton); R.G. Hawley for the MSCV vectors (The George Washington University Medical Center); M. van Lohuizen for the pRETRO-SUPER construct (The Netherlands Cancer Institute) and R. Jaenisch for the DR-4 mouse strain (Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology). This work was mostly supported by a grant from Génome Québec to G.S. and in part by a grant from the Réseau de Recherche en Transgenèse du Québec to J.H. M.B. is a recipient of a Canadian Institutes of Health Research (CIHR) studentship, and G.S. is a recipient of a Canada Research Chair in molecular genetics of stem cells and a scholar of the Leukemia Lymphoma Society of America.
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M.B. performed all the experiments and the analyses described herein, except for I-PCR (S.G.), spectral karyotyping and FISH analyses (J.H.). aCGH experiments and chimeras production were conducted by the services mentioned above. M.B. wrote the manuscript, prepared all the figures and performed the experiments under the guidance of G.S.
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Supplementary information
Supplementary Fig. 1
Generation of retroviral vectors. (PDF 38 kb)
Supplementary Fig. 2
Cre-induced recombination between integrated proviruses. (PDF 252 kb)
Supplementary Fig. 3
Display showing the chromosomal deletions confirmed in ES cells. (PDF 94 kb)
Supplementary Fig. 4
Evaluation of inter-chromosomal recombination events. (PDF 180 kb)
Supplementary Fig. 5
Full-length gels and blots. (PDF 161 kb)
Supplementary Table 1
Summary of the Cre-mediated recombination around 11 randomly chosen loci. (PDF 56 kb)
Supplementary Table 2
In vitro differentiation of primary and tertiary clones carrying deletions. (PDF 8 kb)
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Bilodeau, M., Girard, S., Hébert, J. et al. A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells. Nat Methods 4, 263–268 (2007). https://doi.org/10.1038/nmeth1011
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DOI: https://doi.org/10.1038/nmeth1011
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