睾丸注射法制备携带徐淮山羊过氧化氢酶体激活增殖受体γ基因转基因羊的研究

›› 2013, Vol. 40 ›› Issue (6): 153-157.

睾丸注射法制备携带徐淮山羊过氧化氢酶体激活增殖受体γ基因转基因羊的研究

朱才业¹, 李伟¹, 朱睿¹, 韦光辉¹, 邱峰龙¹, 秦玉蓉², 张亚妮¹, 李碧春¹

1. 扬州大学动物科学与技术学院, 江苏扬州 225009;
2. 南京金斯瑞生物科技有限公司, 江苏南京 210000

收稿日期:2012-11-05 出版日期:2013-06-20 发布日期:2013-06-20
通讯作者: 李碧春 E-mail:yubcli@yzu.edu.cn
作者简介:朱才业(1986-),男,甘肃人,硕士生,研究方向:动物胚胎工程与遗传工程。
基金资助:
国家转基因重大专项(2008ZX08008-003、2009ZX08008-003B)。

Study on Generation of Xuhuai Goat PPARγ Gene Overexpression Transgenic Sheep by Testicular Injection

ZHU Cai-ye¹, LI Wei¹, ZHU Rui¹, WEI Guang-hui¹, QIU Feng-long¹, QIN Yu-rong², ZHANG Ya-ni¹, LI Bi-chun¹

1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China;
2. Gen Script (Nanjing) Co., Ltd., Nanjing 210000, China

Received:2012-11-05 Online:2013-06-20 Published:2013-06-20

摘要/Abstract

摘要： 为探究睾丸注射法制备转基因动物的可能性,将携带有徐淮山羊过氧化氢酶体激活增殖受体γ(PPARγ)和绿色荧光蛋白标签的重组载体经脂质体包裹后,将线性化的重组质粒pEGFP-PPARγ通过随机打点注射入性成熟的湖羊睾丸内,采用常规PCR、基因组DNA点杂交及蛋白免疫印迹技术检测外源PPARγ基因在湖羊体内稳定整合的能力,并检测F1代阳性个体屠宰性能、肌内脂肪含量、器官重量及肉品质。F1代羔羊检测转基因羊阳性率为13.7%,证实外源基因在转基因后代体内成功表达。阳性个体胴体重和屠宰率极显著低于对照组个体(P<0.01),阳性个体骨肉比极显著高于对照组个体(P<0.01);阳性个体背最长肌脂肪含量显著高于对照组个体(P<0.05),腰肌内脂肪含量与股二头肌内脂肪含量差异不显著(P>0.05);阳性个体肺脏和小肠极显著大于对照组个体(P<0.01),阳性个体脾脏显著大于对照组个体(P<0.05),阳性个体心脏和肺脏显著小于对照组个体(P<0.05),肝脏、肾脏、胃、睾丸、皮、头之间差异不显著(P>0.05);阳性个体股二头肌嫩度显著低于对照组个体(P<0.05),pH、肉色、失水率之间差异不显著(P>0.05)。

关键词: 过氧化氢酶体激活增殖受体; 睾丸注射; 转基因羊

Abstract: To explore the possibility of transgenic animals by testicular injection, the Xuhuai goat peroxisome proliferator-activated receptor expression vector pEGFP-PPARγ was using lipofection method, then the linearized recombinant plasmid pEGFP-PPARγ by random dot injected into sexually mature Hu sheep testis. Conventional PCR, genomic DNA dot blotting and Western blotting detection the ability of exogenous PPARγ gene in Hu sheep stable integration. Detection positive individuals and negative individuals of F1 generation of performance of slaughter,the intramuscular fat content, organ weight and meat quality. Positive rate of transgenic sheep was to 13.7%, initially confirmed that the foreign gene PPARγ could be integrated into the genome of Hu sheep and inherited to the next generation. Carcass weight and slaughter rate of positive individuals were significantly lower (P<0.01) than negative individuals in F1 Generation, The proportion of flesh of positive individuals was significantly higher (P<0.01) than negative individuals in F1 Generation;the fat content of longissimus muscle of positive individuals was higher (P<0.05)than negative individuals, the fat content of psoas and biceps femoris were no significant difference (P>0.05);positive individuals lung, small intestine were significantly higher (P<0.01) than negative individuals, positive individuals spleen was higher (P<0.05) than negative individuals, positive individuals heart, lung were lower (P<0.05) than negative individuals, liver, kidney, stomach, testis, skin, and head were no significant difference (P>0.05);positive individuals was lower (P<0.05) than negative individuals, no significant difference between the pH, meat color, water loss rate.

Key words: peroxisome proliferator-activated receptor; testicular injection; transgenic sheep

中图分类号:

S827

朱才业, 李伟, 朱睿, 韦光辉, 邱峰龙, 秦玉蓉, 张亚妮, 李碧春. 睾丸注射法制备携带徐淮山羊过氧化氢酶体激活增殖受体γ基因转基因羊的研究[J]. , 2013, 40(6): 153-157.

ZHU Cai-ye, LI Wei, ZHU Rui, WEI Guang-hui, QIU Feng-long, QIN Yu-rong, ZHANG Ya-ni, LI Bi-chun. Study on Generation of Xuhuai Goat PPARγ Gene Overexpression Transgenic Sheep by Testicular Injection[J]. , 2013, 40(6): 153-157.

参考文献

1 孙敏, 倪黎纲, 施青青, 等. 睾丸注射Mx基因生产抗病转基因鸡初探[J]. 扬州大学学报(农业与生命科学版), 2010, 31(2): 14~18.
2 李想, 逍遥, 张鑫, 等. 转基因动物研究进展[J]. 中国奶牛, 2009, 12: 25~30.
3 李福兵,魏泓,孙新民,等. 睾丸内注射pEGFP-N1体内转染精子并在山羊早期胚胎成功表达[J]. 中国实验动物学报,2005, 13(2): 110~114.
4 沈新明, 乔贵林, 张玲, 等. 用曲细精管微注射法建立绿色荧光蛋白转基因小鼠[J]. 第一军医大学学报, 2002, 22(3): 250~253.
5 肖红卫, 郑新民, 陈思怀, 等. 精子介导生产转hCD59基因猪[J]. 华中农业大学学报, 2006, 25(2): 170~173.
6 林婄婄,高中元,乔利英,等. 核受体因子 PPARγ的研究进展[J]. 四川畜牧兽医,2011(5):32~33.
7 曹阳, 高华颖, 李庆伟. 精子干细胞转染法制备转基因兔的研究[J]. 高技术通讯, 2001, 10: 17~21.
8 黄荣, 闫益波, 王子玉, 等. 精子载体法在转基因家畜中的应用与前景[J]. 家畜生态学报, 2010, 31(6): 87~91.
9 路立里, 郝耿, 於建国, 等. 精子载体法转基因技术研究进展[J]. 中国畜牧兽医, 2010, 37(9): 149~153.
10 Dhup S, Majumdar S S. Transgenesis via permanent integration of genes in repopulating spermatogonial cells in vivo[J]. Na Methods, 2008, 5,601~603.
11 Farre L, Rigau T, Mogas T, et al. Adenovirus-mediated introduction of DNA into pig sperm and offspring[J]. Mol Reprod Dev, 1999, 53(2): 149~158.
12 Giuili G, Tomljenovic A, Labrecque N, et al. Murine spermatogonial stem cells: Targeted transgene expression and purification in an active state[J]. EMBO Rep, 2002, 3(8): 753~759.
13 He X, Li H, Zhou Z, et al. Production of brown/yellow patches in the SLC7A11 transgenic sheep via testicular injection of transgene[J].Genetics and Genomics,2012, 39(6): 281~285.
14 Isabelle I,Stephen G. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators[J]. Nature,1990,347:645~650.
15 Kim J H, Jung-Ha H S, Lee H T, et al. Development of a positive method for male stem cell mediated gene transfer in mouse and pig [J]. Mol Reprod Dev, 1997, 46(4): 515~526.
16 Sato M, Iwase R, Kasai K, et al. Direct injection of foreign DNA into mouse testis as a possible alternative of sperm-mediated gene transfer[J]. Anim Biotech, 1994, 5(1): 19~31.
17 Wall R J. New gene transfer methods[J]. Theriogenology, 2002, 50(1): 189~201.
18 Yamazaki Y, Fujimoto H, Ando H, et al. In vivo gene transfer to mouse spermatogenic cells by deoxyribonucleic acid injection into seminiferous tubules and subsequent electroporation[J]. Biology of Reproduction, 1998, 59: 1439~1444.