A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin

Biology of Reproduction
Satoshi WatanabeAkira Onishi

Abstract

Puromycin N-acetyl transferase gene (pac), of which the gene product catalyzes antibiotic puromycin (an effective inhibitor of protein synthesis), has been widely used as a dominant selection marker in embryonic stem (ES) cell-mediated transgenesis. The present study is the first to report on the usefulness of puromycin for production of enhanced green fluorescent protein (EGFP) transgenic piglets after somatic cell cloning and embryo transfer. Somatic cells isolated from porcine fetuses at 73 days of gestation were immediately electroporated with a transgene (pCAG-EGFPac) carrying both EGFP cDNA and pac. This procedure aims to avoid aging effects thought to be generated during cell culture. The recombinant cells were selected with puromycin at a low concentration (2 microg/ml), cultured for 7 days, and then screened for EGFP expression before somatic cell cloning. The manipulated embryos were transplanted into the oviducts of 14 foster mother sows. Four of the foster sows became pregnant and nine piglets were delivered. Of the nine piglets, eight died shortly after birth and one grew healthy after weaning. Results indicate that puromycin can be used for the selection of recombinant cells from noncultured cells, and moreover, m...Continue Reading

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Citations

Nov 26, 2009·Transgenic Research·Jason W RossRandall S Prather
Aug 31, 2010·Transgenic Research·Jannik E JakobsenJacob Giehm Mikkelsen
Jan 30, 2007·Reproduction, Fertility, and Development·Gábor VajtaZoltán Macháty
Aug 30, 2008·Cloning and Stem Cells·Hitomi MatsunariHiroshi Nagashima
Dec 6, 2007·Genome Biology·Karl J ClarkScott C Fahrenkrug
Mar 23, 2011·The Journal of Reproduction and Development·Koji Yoshioka
Feb 26, 2008·The Journal of Reproduction and Development·Mayuko KuromeHiroshi Nagashima
Dec 25, 2008·The Journal of Reproduction and Development·Seong-Keun ChoJin-Hoi Kim
Jul 3, 2009·The Journal of Reproduction and Development·Hitomi Matsunari, Hiroshi Nagashima
Nov 5, 2011·Yi chuan = Hereditas·Qing-Ran KongZhong-Hua Liu
Mar 6, 2009·Reproduction : the Official Journal of the Society for the Study of Fertility·Yue-Mao ZhengHui Lin
Mar 15, 2016·Frontiers in Genetics·Lawrence B SchookTiago Collares
Mar 6, 2015·The Journal of Reproduction and Development·Masahito WatanabeHiroshi Nagashima
Jun 20, 2008·Reproduction in Domestic Animals = Zuchthygiene·J LiG Vajta
Apr 7, 2016·Molecular Biotechnology·Bartosz F GrześkowiakAnna Woźniak
Jun 19, 2012·Cell Stem Cell·Shunichi SuzukiAkira Onishi
Aug 9, 2006·Molecular Reproduction and Development·Satoshi WatanabeMasahiro Sato
Aug 29, 2019·In Vitro Cellular & Developmental Biology. Animal·Rui WangXiang-Xing Zhu

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Methods Mentioned

BETA
transgenic
transfection
PCR
electrophoresis
Assay
transfections
biopsy

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