N-乙酰嘌呤霉素转移酶基因（pac）的产物对嘌呤霉素起催化作用，该基因作为胚胎干细胞（ES）介导的基因转移中的一个显性选择标记已得到广泛应用。本研究是第一次报道在体细胞克隆和胚胎移植后成功应用嘌呤霉素标记获得了转强化型绿色荧光蛋白（EGFP）基因的小猪。从妊娠73天的猪胎儿身上分离得到体细胞后，立即用携带EGFP cDNA和pac的空载体（pCAG-EGFPac）进行电穿孔基因转移处理。该操作的目的在于避免细胞培养中可能存在的年龄效应。重组的细胞用低浓度（2 µg/ml）的嘌呤霉素进行筛选，然后培养7天，在体细胞克隆前对EGFP的表达进行显影。处理过的胚胎移入14头代孕母猪的输卵管内。有4头代孕母猪妊娠并产下了9头小猪。9头小猪中的8头在出生后很快死亡，1头健康地成长到断乳。结果表明嘌呤霉素可被用于从非培养细胞中筛选重组细胞，而且还可以作为通过核移植技术获得遗传改造小猪的参照标记。
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http://www.biolreprod.org/cgi/content/full/72/2/309
http://www.biolreprod.org/cgi/content/abstract/biolreprod.104.031591v1
http://www.bioone.org/perlserv/?request=get-abstract&doi=10.1095%2Fbiolreprod.104.031591
英文原文：BOR - Papers in Press, published online ahead of print September 22, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.031591
BIOLOGY OF REPRODUCTION 72, 309–315 (2005)
DOI: 10.1095/biolreprod.104.031591
© 2005 by the Society for the Study of Reproduction, Inc.
A Novel Method for the Production of Transgenic Cloned Pigs: Electroporation-Mediated Gene Transfer to Non-Cultured Cells and Subsequent Selection with Puromycin1
Satoshi Watanabe2, Masaki Iwamoto4,5, Shun-ichi Suzuki4, Daiichiro Fuchimoto4, Daisuke Honma4, Takashi Nagai3,4, Michiko Hashimoto4,5, Satoko Yazaki4,5, Masahiro Sato6 and Akira Onishi4
Department of Developmental Biology,4 Division of Insect and Animal, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-0901, Japan Prime Tech Ltd.,5 Tsuchiura, Ibaraki, 300-841, Japan The Institute of Medical Sciences,6 Tokai University, Bohseidai, Isehara, Kanagawa, 259-1143, Japan
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 µg/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. 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, may confer the production of genetically engineered newborns via nuclear transfer techniques in pigs.
developmental biology, early development, embryo, gene regulation
