N-乙酰嘌呤霉素轉移酶基因（pac）的產物對嘌呤霉素起催化作用，該基因作為胚胎干細胞（ES）介導的基因轉移中的一個顯性選擇標記已得到廣泛應用,。本研究是第一次報道在體細胞克隆和胚胎移植后成功應用嘌呤霉素標記獲得了轉強化型綠色熒光蛋白（EGFP）基因的小豬,。從妊娠73天的豬胎兒身上分離得到體細胞后,，立即用攜帶EGFP cDNA和pac的空載體（pCAG-EGFPac）進行電穿孔基因轉移處理。該操作的目的在于避免細胞培養(yǎng)中可能存在的年齡效應,。重組的細胞用低濃度（2 µg/ml）的嘌呤霉素進行篩選,，然后培養(yǎng)7天,，在體細胞克隆前對EGFP的表達進行顯影。處理過的胚胎移入14頭代孕母豬的輸卵管內,。有4頭代孕母豬妊娠并產下了9頭小豬,。9頭小豬中的8頭在出生后很快死亡，1頭健康地成長到斷乳,。結果表明嘌呤霉素可被用于從非培養(yǎng)細胞中篩選重組細胞,，而且還可以作為通過核移植技術獲得遺傳改造小豬的參照標記。
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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
