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Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons

Abstract

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection into the pronuclei of fertilized oocytes of synthetic mRNA encoding the SB100X hyperactive transposase together with plasmid DNA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNA is followed by enzyme-mediated excision of the transgene cassette from the plasmids and its permanent genomic insertion to produce stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes 2 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies (numbers of transgenic founders obtained per injected embryo) to lentiviral approaches, without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.

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Figure 1: Application of Sleeping Beauty transposons for gene delivery.
Figure 2: In vitro mRNA synthesis.
Figure 3: Timelines for rabbit manipulations.
Figure 4: Overview of rabbit transgenesis.
Figure 5: Identification of transgene integration by PCR.

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Zixuan Zhao, Xinyi Chen, … Hanry Yu

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Acknowledgements

Z.B. was supported by the Hungarian grants OTKA NK 104397 and NN 108921. The research of V.L. was supported by grant TA02010013 from the Technological Agency of the Czech Republic and grant LH12061 from the Ministry of Education, Youth and Sports of the Czech Republic. Financial support from grants of the Deutsche Forschungsgemeinschaft (IV 21/6-1) to Z. Ivics, as well as from the Austrian Genome Research Programme GEN-AU II and III (Austromouse) to T.R., is gratefully acknowledged.

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Authors and Affiliations

Authors

Contributions

Z. Izsvák, Z. Ivics, Z.B. and L.H. designed the research; O.I.H., L.H., L.M., T.Y.Y., S.B., V.Z., M.P. and T.R. performed the experiments; Z. Ivics, A.G. and Z. Izsvák analyzed the data; and Z. Ivics, Z.B. and L.H. wrote the manuscript.

Corresponding authors

Correspondence to Zoltán Ivics, László Hiripi, Zsuzsanna Bösze or Zsuzsanna Izsvák.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 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

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

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Ivics, Z., Hiripi, L., Hoffmann, O. et al. Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons. Nat Protoc 9, 794–809 (2014). https://doi.org/10.1038/nprot.2014.009

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