Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer
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The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2 % of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.
KeywordspiggyBac Transposon Transposase SCNT Cloning Transgenesis
This study was supported by a grant from the National Science Foundation for Young Scholars of China (Grant Number: 31101689), a grant from the National High Technology Research and Development Program of China (863 Program, Grant Number: 2011AA100304), a grant from Department of Science and Technology of Guangdong, China (Grant Number: 2011A020901001) and by National Institutes of Health Grants 5P20RR024206 and R01 GM083158-01A1 (to S.M.).
- Al-Mashhadi RH, Sorensen CB, Kragh PM, Christoffersen C, Mortensen MB, Tolbod LP, Thim T, Du Y, Li J, Liu Y, Moldt B, Schmidt M, Vajta G, Larsen T, Purup S, Bolund L, Nielsen LB, Callesen H, Falk E, Mikkelsen JG, Bentzon JF (2013) Familial hypercholesterolemia and atherosclerosis in cloned minipigs created by DNA transposition of a human PCSK9 gain-of-function mutant. Sci Transl Med 5(166):166ra161. doi: 10.1126/scitranslmed.3004853 CrossRefGoogle Scholar
- Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, Enos J, Forsythe T, Golueke P, Jurgella G, Koppang R, Lesmeister T, Mallon K, Mell G, Misica P, Pace M, Pfister-Genskow M, Strelchenko N, Voelker G, Watt S, Thompson S, Bishop M (2000) Production of cloned pigs from in vitro systems. Nat Biotechnol 18(10):1055–1059. doi: 10.1038/80242 CrossRefPubMedGoogle Scholar
- Deng W, Yang D, Zhao B, Ouyang Z, Song J, Fan N, Liu Z, Zhao Y, Wu Q, Nashun B, Tang J, Wu Z, Gu W, Lai L (2011) Use of the 2A peptide for generation of multi-transgenic pigs through a single round of nuclear transfer. PLoS One 6(5):e19986. doi: 10.1371/journal.pone.0019986 CrossRefPubMedGoogle Scholar
- Garcia-Vazquez FA, Ruiz S, Matas C, Izquierdo-Rico MJ, Grullon LA, De Ondiz A, Vieira L, Aviles-Lopez K, Gutierrez-Adan A, Gadea J (2010) Production of transgenic piglets using ICSI-sperm-mediated gene transfer in combination with recombinase RecA. Reproduction 140(2):259–272. doi: 10.1530/REP-10-0129 CrossRefPubMedGoogle Scholar
- Jakobsen JE, Li J, Kragh PM, Moldt B, Lin L, Liu Y, Schmidt M, Winther KD, Schyth BD, Holm IE, Vajta G, Bolund L, Callesen H, Jorgensen AL, Nielsen AL, Mikkelsen JG (2011) Pig transgenesis by Sleeping Beauty DNA transposition. Transgenic Res 20(3):533–545. doi: 10.1007/s11248-010-9438-x CrossRefPubMedGoogle Scholar
- Marh J, Stoytcheva Z, Urschitz J, Sugawara A, Yamashiro H, Owens JB, Stoytchev I, Pelczar P, Yanagimachi R, Moisyadi S (2012) Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis. Proc Natl Acad Sci USA 109(47):19184–19189. doi: 10.1073/pnas.1216473109 CrossRefPubMedGoogle Scholar
- Staunstrup NH, Madsen J, Primo MN, Li J, Liu Y, Kragh PM, Li R, Schmidt M, Purup S, Dagnaes-Hansen F, Svensson L, Petersen TK, Callesen H, Bolund L, Mikkelsen JG (2012) Development of transgenic cloned pig models of skin inflammation by DNA transposon-directed ectopic expression of human beta1 and alpha2 integrin. PLoS One 7(5):e36658. doi: 10.1371/journal.pone.0036658 CrossRefPubMedGoogle Scholar
- Urschitz J, Kawasumi M, Owens J, Morozumi K, Yamashiro H, Stoytchev I, Marh J, Dee JA, Kawamoto K, Coates CJ, Kaminski JM, Pelczar P, Yanagimachi R, Moisyadi S (2010) Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects. Proc Natl Acad Sci USA 107(18):8117–8122. doi: 10.1073/pnas.1003674107 CrossRefPubMedGoogle Scholar