Abstract
Recently, we established the Sleeping Beauty transposon system for germ line competent transgenesis in the pig. Here, we extend this approach to re-target a transposon-tagged locus for a site-specific gene knock-in, and generated a syngeneic cohort of piglets carrying either the original transposon or the re-targeted event. A Cre-loxP-mediated cassette exchange of the tagging transposon with a different reporter gene was performed, followed by flow cytometric sorting and somatic cell nuclear transfer of recombined cells. In parallel, the original cells were employed in somatic cell nuclear transfer to generate clone siblings, thereby resulting in a clone cohort of piglets carrying different reporter transposons at an identical chromosomal location. Importantly, this strategy supersedes the need for an antibiotic selection marker. This approach expands the arsenal of genome engineering technologies in domestic animals, and will facilitate the development of large animal models for human diseases. Potentially, the syngeneic cohort of pigs will be instrumental for vital tracking of transplanted cells in pre-clinical assessments of novel cell therapies.
This is a preview of subscription content, log in via an institution to check access.
Abbreviations
- BLAST:
-
Basic local alignment search tool
- CPI:
-
Cytoplasmic injection of plasmids
- FACS:
-
Fluorescence activated cell sorting
- HR:
-
Homologous recombination
- LED:
-
Light emitting diode
- PNI:
-
Pronuclear injection (of zygote)
- RMCE:
-
Recombinase mediated cassette exchange
- SB:
-
Sleeping Beauty (transposon system)
- SCNT:
-
Somatic cell nuclear transfer
- TALEN:
-
Transcription activator-like element nuclease
- ZFN:
-
Zinc finger nuclease
References
Al-Mashhadi RH, Sørensen CB, Kragh PM, Christoffersen C et al (2013) Familial hypercholesterolemia and atherosclerosis in cloned minipigs created by DNA transposition of a human PCSK9 gain-of-function mutant. Sci Transl Med 5:166ra1
Bosch P, Forcato DO, Alustiza FE, Alessio AP et al (2015) Exogenous enzymes upgrades transgenesis and genetic engineering in livestock. Cell Mol Life Sci 72:1907–1929
Esteban MA, Xu J, Yang J, Peng M, Qin D et al (2009) Generation of induced pluripotent stem cell lines from tibetan miniature pig. J Biol Chem 284:17634–17640
Ezashi T, Telugu BP, Alexenko AP, Sachdev S, Sinha S et al (2009) Derivation of induced pluripotent stem cells from pig somatic cells. Proc Natl Acad Sci USA 106:10993–10998
Garrels W, Mates L, Holler S, Dalda A et al (2011) Germline transgenic pigs by Sleeping Beauty transposition in porcine zygotes ad targeted integration in the pig genome. PLoS One 6:e23573
Garrels W, Holler S, Cleve N, Niemann N et al (2012) Assessment of fecundity and germ line transmission in two transgenic pig lines produced by Sleeping Beauty transposition. Genes 3:615–633
Garrels W, Holler D, Taylor U, Herrmann D et al (2014) Assessment of fetal cell chimerism in transgenic pig lines generated by Sleeping Beauty transposition. PLoS One 8:e96673
Hai T, Teng F, Guo R, Zhou Q (2014) One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 24:372–375
Hauschild J, Petersen B, Santiago Y, Queisser AL et al (2011) Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proc Natl Acad Sci USA 108:12013–12017
Holker M, Petersen B, Hassel P, Kues WA et al (2005) Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos. Cloning Stem Cells 7:35–44
Hsiao FS, Lian WS, Lin SP, Lin CJ, Lin YS et al (2011) Toward an ideal animal model to trace donor cell fates after stem cell therapy: production of stably labeled multipotent mesenchymal stem cells from bone marrow of transgenic pigs harboring enhanced green fluorescence protein gene. J Anim Sci 89:3460–3472
Ivics Z, Garrels W, Mates L, Yau TY et al (2014) Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons. Nat Protoc 9:810–827
Jakobsen JE, Johansen MG, Schmidt M, Dagnaes-Hansen F et al (2013) Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT). Transgenic Res 22:709–723
Li X, Yang Y, Bu L, Guo X et al (2014) Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing. Cell Res 24:501–504
Mátés L, Chuah MK, Belay E, Jerchow B et al (2009) Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet 41:753–761
Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS et al (2008) Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 321:1837–1841
Shigeta T, Hsu HC, Enosawa S, Matsuno N et al (2013) Transgenic pig expressing the red fluorescent protein kusabira-orange as a novel tool for preclinical studies on hepatocyte transplantation. Transplant Proc 45:1808–1810
Tasic B, Hippenmeyer S, Wang C, Gamboa M et al (2011) Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc Natl Acad Sci USA 108:7902–7907
Whyte JJ, Prather RS (2011) Genetic modifications of pigs for medicine and agriculture. Mol Reprod Dev 78:879–891
Acknowledgments
The flow cytometric sorting was performed at the Core Facility Cell Sorting of the Medical School Hannover (MHH) by M. Ballmeier. H.J. Schuberth (Veterinary University of Hannover) gratefully supported the FACS analysis. The microsatellite analysis was performed by Annett Weigend and Natalie Janus (Department of Breeding and Genetic Resource, FLI). I. Ott, P. Hassel, L. Schindler, M. Ziegler, R. Poppenga, E. Kuhfeld, T. Peker, and J. Kun are gratefully acknowledged for excellent technical assistance. This work was supported by grants of the Deutsche Forschungsgemeinschaft (KU 1586/2-1 and IV 21/6-1), and by International Fellowships of ICAR to AM and TRT, respectively. AM is enrolled at the Hannover Graduate School for Veterinary Pathobiology, Neuroinfectiology and Translational Medicine (HGNI), and this work represents a partial fulfilment of the requirements for the degree of a Ph.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflict of interests.
Additional information
Wiebke Garrels and Ayan Mukherjee have contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Garrels, W., Mukherjee, A., Holler, S. et al. Identification and re-addressing of a transcriptionally permissive locus in the porcine genome. Transgenic Res 25, 63–70 (2016). https://doi.org/10.1007/s11248-015-9914-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-015-9914-4