体细胞核移植技术生产猪植酸酶转基因胚胎的研究

›› 2014, Vol. 41 ›› Issue (11): 1-6.

• 生物技术 • 下一篇

体细胞核移植技术生产猪植酸酶转基因胚胎的研究

戴建军^1,2, 吴彩凤^1,2, 张树山^1,2, 顾晓龙^1,2, 张廷宇^1,2, 吴志强^1,2, 张德福^1,2

1. 上海市农业科学院畜牧兽医研究所, 上海 201106;
2. 上海农业遗传育种重点实验室动物遗传工程研究室, 上海 201106

收稿日期:2014-04-28 出版日期:2014-11-20 发布日期:2014-12-06
通讯作者: 张德福 E-mail:zhangdefuzdf@163.com
作者简介:戴建军(1979-),男,江苏人,硕士,助理研究员,研究方向:动物胚胎工程。
基金资助:
国家自然科学基金(31372315);国家转基因重大专项(2013ZX08006-005);上海市科委农业成果转化项目(123919N0700、1333919N1700)。

Study on the Production of Porcine Phytase Transgenic Embryos with Somatic Cell Nuclear Transfer

DAI Jian-jun^1,2, WU Cai-feng^1,2, ZHANG Shu-shan^1,2, GU Xiao-long^1,2, ZHANG Ting-yu^1,2, WU Zhi-qiang^1,2, ZHANG De-fu^1,2

1. Institute of Animal Science and Veterinary Medicine, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China;
2. Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-genetics and Breeding, Shanghai 201106, China

Received:2014-04-28 Online:2014-11-20 Published:2014-12-06

摘要/Abstract

摘要： 为获得具有植酸酶腮腺特异性表达的猪转基因克隆胚胎,本研究使用植酸酶腮腺特异性表达的DNA质粒(包含腮腺分泌蛋白(parotid secretary protein,PSP)启动子与终止子序列、Neo筛选基因、绿色荧光蛋白(EGFP)报告基因和高比活的植酸酶appA基因),采用脂质体转染和基因素418(G418)药物抗性筛选的方法获取稳转细胞系,并利用体细胞核移植技术获得植酸酶转基因胚胎。结果表明,本研究构建的DNA质粒可用于细胞筛选,且质粒越小,细胞的转染效率越高,14.89 kb的YM6552仅获得了7.1%的转染率,EGFP质粒则获得了43.4%的转染效率。在单克隆形成上,较小的pYN3600也获得了更高的单克隆形成数(25个),其中表达EGFP的单克隆有14个,植酸酶PCR阳性集落有11个,高于YM6552的单克隆数(19、8和6)。转基因细胞构建重构胚胎后,所有的胚胎均能表达绿色荧光蛋白,虽其体外发育能力有所下降,但差异不显著(P>0.05)。综上所述,本研究所采用的植酸酶质粒、细胞筛选方法和核移植技术可生产植酸酶重构胚。

关键词: 植酸酶; 腮腺分泌蛋白; 转基因核移植; 猪

Abstract: In order to get porcine phytase salivary glands specific express cloned embryos,this study used phytase specific expressed plasmid DNA (including the promotor and terminater sequences of parotid secretary protein (PSP) gene,Neo selected gene and EGFP report gene and high enzymatic activity phytase gene (appA)) and transfected it into porcine fetal fibroblast cells with liposome.The stably transfected cell lines were screened by G418 selection,and positive phytase transgenic embryos were constructured by somatic cell nuclear transfer.The results showed that the phytase plasmid of this experiment could be used to carry on the cell selection,and the more smaller of plasmid DNA size,the more higher transfected efficiency was got.The plasmid YM6552 with 14.89 kb size only got 7.1% transfected efficiency,while EGPF plasmid got 43.4%.In the formation of single cell colony,pYN3600 (more smaller DNA size) got 25 colony numbers,and 14 of them expressed green fluorescent protein,11 of them were phytase PCR positive.They were much higher than those of YM6552 (19,8 and 6).All the reconstructed transgenic embryoes from the positive cell lines were expressed green fluorescent protein.Though the in vitro development ability of transgenic cloned embryos were a little lower than that of control group,there were no significant differences between them (P>0.05).In conclusion,all the phytase plasmid,cell selection method and nuclear transfer techniques could be used to produce porcine phytase salivary glands specific express embryos.

Key words: phytase; parotid secretary protein; transgenic nuclear transfer; porcine

中图分类号:

Q813

戴建军, 吴彩凤, 张树山, 顾晓龙, 张廷宇, 吴志强, 张德福. 体细胞核移植技术生产猪植酸酶转基因胚胎的研究[J]. , 2014, 41(11): 1-6.

DAI Jian-jun, WU Cai-feng, ZHANG Shu-shan, GU Xiao-long, ZHANG Ting-yu, WU Zhi-qiang, ZHANG De-fu. Study on the Production of Porcine Phytase Transgenic Embryos with Somatic Cell Nuclear Transfer[J]. , 2014, 41(11): 1-6.

参考文献

1 尹海芳,樊宝良,赵志辉,等.猪腮腺分泌蛋白基因序列和蛋白结构比较分析及组织表达谱鉴定[J].科学通报,2003,48(13):1432～1437.
2 田兴华,樊宝良,李宁,等.猪腮腺分泌蛋白在腮腺生后发育过程中表达特性研究[J].中国畜牧杂志,2005a,41(10):15～17.
3 田兴华.猪腮腺分泌蛋白表达特性及表达调控元件的研究[D].北京:中国农业大学,2005b.
4 齐丽娜.人α-唾液腺淀粉酶基因和广西巴马小型猪腮腺分泌蛋白基因 5'调控序列的克隆及功能验证[D].南宁:广西大学,2012.
5 李紫聪,张茂,许惠,等.转β-甘露聚糖酶基因 manA小鼠的制备[J].广东农业科学,2013,40(14):144～146.
6 郭江,吕林,李素芬,等.转植酸酶基因玉米对肉仔鸡血液生理生化指标和免疫功能的影响[J].中国畜牧兽医,2012,39(12):93～96.
7 Golovan S P,Meidinger R G,Ajakaiye A,et al.Pigs expressing salivary phytase produce low-phosphorus manure[J].Nature Biotechnology,2001a,19(8):741～745.
8 Golovan S P,Hayes M A,Phillips J P,et al.Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control[J].Nat Biotechnol,2001b,19(5):429～433.
9 Guan L Z,Sun Y P,Xi Q Y,et al.β-glucanase specific expression in the parotid gland of transgenic mice[J].Transgenic Res,2013,22:805～812.
10 Huang M R,Li Z C,Huang X L,et al.Co-expression of two fibrolytic enzyme genes in CHO cells and transgenic mice[J].Transgenic Res,2013,22(4):779～790.
11 Kreiss P,Cameron B,Rangara R,et al.Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency[J].Nucleic Acids Res,1999,27(19):3792～3798.
12 Kobelt D,Schleef M,Schmeer M,et al.Performance of high quality minicircle DNA for in vitro and in vivo gene transfer[J].Mol Biotechnol,2013,53(1):80～89.
13 Laursen J,Krogh-Pedersen H,Dagnaes-Hansen F,et al.The main regulatory region in the murine PSP gene is a parotid gland enhancer[J].Transgenic Res,1998,7(6):413～420.
14 Lee G S,Kim H S,Hyun S H,et al.Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein[J].Theriogenology,2005,63(4):973～991.
15 Li Q,Wei H,Guo Y,et al.Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer[J].Progress in Nat Sci,2009,19(6):699～704.
16 Liu D W,Zhang M,Li Z C,et al.Generation of transgenic mice producing fungal xylanase in the saliva as a model for improving feed digestibility[J].Afric J Bioechnol,2012,11(94):16112～16117.
17 Svendsen P,Laursen J,Krogh-Pedersen H,et al.Novel salivary gland specific binding elements located in the PSP proximal enhancer core[J].Nucleic Acids Res,1998,26(11):2761～2770.
18 Yin H F,Zhao Z H,Fan B L,et al.cDNA cloning,genomic structure,chromosomal mapping,and expression analysis of parotid secretory protein in pig[J].Genomics,2004,83:9～18.
19 Yin W X,Xiang P,Li Q L.Investigations of the effect of DNA size in transient transfection assay using dual luciferase system[J].Anal Biochem,2005,346(2):289～294.
20 Yin H F,Fan B L,Yang B,et al.Cloning of pig parotid secretory protein gene upstream promoter and the establishment of a transgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control[J].J Anim Sci,2006,84(3):513～519.
21 Zakhartchenko V,Mueller S,Alberio R,et al.Nuclear transfer in cattle with non-transfected and transfected fetal or cloned transgenic fetal and postnatal fibroblasts[J].Mol Reprod Dev,2001,60(3):362～369.
22 Zhang T Y,Dai J J,Wu C F,et al.Positive effects of treatment of donor cells with aphidicolin on the preimplantation development of somatic cell nuclear transfer embryos in Chinese Bama mini-pig (Sus scrofa)[J].Anim Sci J,2012,83(2):103～110.

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体细胞核移植技术生产猪植酸酶转基因胚胎的研究

Study on the Production of Porcine Phytase Transgenic Embryos with Somatic Cell Nuclear Transfer

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