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Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

Nature Protocols volume 9, pages 810–827 (2014)Cite this article

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Abstract

The pig has emerged as an important large animal model in biomedical and pharmaceutical research. We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase, together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer (SCNT), and it offers comparable efficacies to lentiviral approaches, without limitations on vector design, issues of transgene silencing and the toxicity and biosafety concerns of working with viral vectors. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in 1 d; generation of germline-transgenic lines by using this protocol takes ∼1 year.

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Figure 1: Application of Sleeping Beauty transposons for gene delivery.
Figure 2: Injection of circular transposon plasmids into the cytoplasm of porcine zygotes.
Figure 3: Timeline flowchart for animal treatment and cytoplasmic injection.
Figure 4: Isolation of porcine zygotes from the oviduct.
Figure 5: Loading of transfer straw with embryos and embryo transfer.
Figure 6: Embryo transfer of microinjected zygotes to synchronized surrogates.
Figure 7: Live imaging of piglets transgenic for a Venus-tagged SB transposon.

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Acknowledgements

This work was financially supported by grants of the Deutsche Forschungsgemeinschaft to W.A.K. and Z. Ivics (KU 1586/2-1 and IV 21/6-1). The expert support and critical input by J.W. Carnwath, S. Holler, B. Barg-Kues, N. Cleve, M. Ziegler and M. Diederich are also gratefully acknowledged. Whole-animal images under fluorescence excitation were done by the Friedrich Loeffler Institute's photographer D.B., and the video was recorded by P. Köhler.

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Author notes

  1. Zoltán Ivics and Wiebke Garrels: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Medical Biotechnology, Paul Ehrlich Institute, Langen, Germany

    Zoltán Ivics

  2. Friedrich-Loeffler-Institut, Institut für Nutztiergenetik, Neustadt, Germany

    Wiebke Garrels & Wilfried A Kues

  3. Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary

    Lajos Mátés

  4. Institute of Laboratory Animal Science, University of Veterinary Medicine Vienna, Vienna, Austria

    Tien Yin Yau & Thomas Rülicke

  5. Max Delbrück Center for Molecular Medicine, Berlin, Germany

    Sanum Bashir & Zsuzsanna Izsvák

  6. Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

    Vaclav Zidek, Vladimír Landa & Michal Pravenec

  7. Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

    Aron Geurts

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Contributions

W.A.K., T.R., M.P., A.G., Z. Ivics and Z. Izsvák designed the study; W.G., L.M., T.Y.Y., S.B., V.Z., V.L., A.G., Z. Ivics and W.A.K. performed the experiments; W.A.K., Z. Ivics and W.G. evaluated the data; and W.A.K., Z. Ivics, T.R., W.G. and Z. Izsvák wrote the manuscript.

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Correspondence to Zoltán Ivics, Wilfried A Kues or Zsuzsanna Izsvák.

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Integrated supplementary information

Supplementary Figure 1 Identification of genomic transgene integration by PCR.

(a) Identification of integrated transposon sequences from mouse genomic DNA samples by PCR with primers that amplify the left ITR of SB. Lanes: 1) H2O; 2) mouse genomic DNA, negative control; 3) mouse genomic DNA, positive control; 4) transgenic founder #1; 5-9) F1 offspring of transgenic founder #1; 10) transgenic founder #2; 11-13) F1 offspring of transgenic founder #2; M) 100-bp molecular size marker. (b) Ouline of the LMPCR procedure. Digestion of genomic DNA with the frequently cutting restriction enzymes BfaI and DpnII and ligation of linkers with a known sequence allows for specific LMPCR amplification of transposon/genomic DNA junctions using primers specific to the transposon ITR (blue arrows) and the linkers (green arrows). GOI, gene of interest; ITR, inverted terminal repeat. (c) Agarose gel with genomic DNA samples. Lanes 1-4) genomic DNA samples of rat founders, 500 ng each; M) DNA size marker. (d) Agarose gel with genomic DNA samples digested with BfaI restriction endonuclease. Lanes 1-4) BfaI-digested DNA samples of rat founders, 200 ng each; M) DNA size marker. (e) Agarose gel with LMPCR products. Lanes 1-4) result of nested PCR following BfaI linker ligation; M) DNA size marker.

Supplementary Figure 2 Locus-specific PCR test of a rat founder and its F1 descendants.

The founder of these F1 animals carried three SB integrations (in chr2, chr4 and chr16), which were transmitted to 13 descendants in different combinations. M, DNA size marker.

Supplementary information

Supplementary Figure 1

Identification of genomic transgene integration by PCR. (PDF 488 kb)

Supplementary Figure 2

Locus-specific PCR test of a rat founder and its F1 descendants. (PDF 361 kb)

Copy number-dependent fluorescence in F2-generation piglets.

The F2 litter of piglets are derived from the crossbreeding of two lines. Each founder carried three monomeric Venus-transposons, which segregated individually during meiosis. The piglets shown carry 0 to 5 copies of the Venus-transposon, and the fluorescent intensity correlates directly with the genotype. The “bluish” piglet in the back is a non-transgenic littermate. (MPG 1838 kb)

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Ivics, Z., Garrels, W., Mátés, L. et al. Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons. Nat Protoc 9, 810–827 (2014). https://doi.org/10.1038/nprot.2014.010

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