Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer
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ORIGINAL PAPER
Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer Zhenfang Wu • Zhiqian Xu • Xian Zou • Fang Zeng • Junsong Shi • Dewu Liu • Johann Urschitz • Stefan Moisyadi • Zicong Li
Received: 1 November 2012 / Accepted: 19 June 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract 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 Z. Wu Z. Xu X. Zou F. Zeng Z. Li (&) Department of Animal Genetics, Breeding and Reproduction, South China Agricultural University, The New Building of College of Animal Science, Room 315, Guangzhou 510642, Guangdong, People’s Republic of China e-mail: [email protected] J. Shi Wen’s Research Institute, Guangdong Wen’s Food Group Co. Ltd., Yunfu 527439, Guangdong, People’s Republic of China D. Liu Department of Animal Production, College of Animal Science, South China Agricultural University, Guangzhou 510642, Guangdong, People’s Republic of China J. Urschitz S. Moisyadi (&) Department of Anatomy, Biochemistry and Physiology, Institute for Biogenesis Research, John A. Burns School of Medicine, University of Hawaii at Manoa, E-124, 1960 East–West Rd., Honolulu, HI 96822, USA e-mail: [email protected]
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. Keywords piggyBac Transposon Transposase SCNT Cloning Transgenesis
Introduction A transgenic animal can be described as one whose genome has been altered by the introduction of foreign genetic material. The term transgenic was introduced by the developers of the initial pronuclear microinjection (PNI) procedure, a method whereby linear transgene DNA is injected into the male pronucleus of a zygote (Gordon et al. 1980). Several alternative procedures for animal transgenesis have been developed in the last 32 years with the aim of improving the efficiency and ease of delivery for the transgenic DNA (Marh et al. 2012; Lavitrano et al. 1989; Lois et al. 2002; Perry et al. 1999; Urschitz et al. 2010). However, the preferred and most widely used method to generate transgenic animals is PNI, due to ease of PNI’s implementation and the lack of specialized requirements such as containment facilities necessary
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Transgenic Res
for lentiviral mediated transgenesis (Lois et al. 2002). However, PNI is impeded by low efficiency, concatemerized transgene i
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