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Production of avian retroviruses and tissue-specific somatic retroviral gene transfer in vivo using the RCAS/TVA system

Nature Protocols volume 7, pages 1167–1183 (2012)Cite this article

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Abstract

Spatiotemporal retroviral gene transfer into specific somatic mammalian cells using the avian RCAS (replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor)/tumor virus A (TVA) system is a versatile tool for performing lineage tracing and gene function analysis in vivo. RCAS retroviruses carrying the subgroup A envelope transduce only genetically engineered mammalian cells that express the cognate avian retroviral receptor TVA. The RCAS/TVA gene delivery system has been successfully used in various different mouse TVA-expression models. This protocol contains a detailed description of the production of high-titer RCAS retroviruses in chicken fibroblasts and the transduction of proliferating TVA-positive somatic mammalian cells in vivo. By taking advantage of the combination of the RCAS/TVA with the 'universal' Cre/loxP system, the protocol can be used in nearly every proliferating cell type in vivo. The protocol takes 4 weeks from transfection of chicken fibroblasts, which act as the host cells for viral production, to the transduction of TVA-transgenic mice.

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Figure 1: Schematic illustration of the principle and different steps of the protocol.
Figure 2: Ectopic expression of the TVA receptor in the pancreas renders mouse cells susceptible to retroviral RCASBP(A)-mediated somatic gene transfer in vivo.
Figure 3: Ubiquitous ectopic expression of the TVA receptor renders different mouse tissue types susceptible to retroviral RCASBP(A)-mediated somatic gene transfer in vivo.

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Acknowledgements

We would like to thank H. Nakhai (Technical University Munich) and S. O'Gorman (Case Western Reserve University) for providing transgenic animals. We are grateful to N. Proudfoot (University of Oxford), P. Bates (University of Pennsylvania), S. Orsulic (Harvard Medical School) and S.H. Hughes (US National Cancer Institute) for providing vectors. This work was supported by funding from Deutsche Krebshilfe (no. 108985 to D.S.). Research was conducted in compliance with the European guidelines for the care and use of laboratory animals and approved by the local authorities.

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  1. Alexander von Werder and Barbara Seidler: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine 2, Technische Universität München, München, Germany

    Alexander von Werder, Barbara Seidler, Roland M Schmid, Günter Schneider & Dieter Saur

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Contributions

A.v.W., B.S. and D.S. performed research and developed the protocol; A.v.W., B.S., R.M.S., G.S. and D.S. wrote the manuscript.

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Correspondence to Dieter Saur.

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Supplementary information

Supplementary Figure 1

Scheme of the wild type Rosa26 (R26) locus and the targeted R26 locus with the integrated lox-stop-lox (LSL) silenced Tva-IRES-LacZnls cassette. Copyright (2008) National Academy of Sciences, U.S.A. Reproduced with permission from Seidler, B. et al. A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors. Proc Natl Acad Sci U S A 105, 10137-10142 (2008). (A) Upper panel: Scheme of the R26 wild type locus. Primer binding sites for genotyping of the R26 wild type locus are depicted. Lower panel: Targeted Rosa26 locus. Black arrowheads flanking the transcriptional stop cassette represent loxP sites. Primer binding sites are indicated by arrows. SA: Splice acceptor. ATG: ATG start codon of TVA. (B-E): Genotyping PCR. (B) PCR analysis of the wild type (WT) and the targeted LSL-R26Tva-lacZ allele. (C) PCR for detection of TVA cDNA or genomic DNA in the mouse, regardless of the TVA strain. (D) PCR to test integrity of the lox-stop-lox (LSL) cassette before Cre mediated recombination. (E) PCR to test Cre mediated deletion of the LSL cassette. Sizes of wild-type and mutant PCR products are indicated. (DOC 113 kb)

Supplementary Method 1

Genotyping protocol for LSL-R26Tva-lacZ/+ , Ptf1aCre/+ and Prm-Cre animals (DOC 57 kb)

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von Werder, A., Seidler, B., Schmid, R. et al. Production of avian retroviruses and tissue-specific somatic retroviral gene transfer in vivo using the RCAS/TVA system. Nat Protoc 7, 1167–1183 (2012). https://doi.org/10.1038/nprot.2012.060

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