Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (17): 3695-3703.doi: 10.3864/j.issn.0578-1752.2013.17.019

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Cloning of Xuhuai Goat SCD1 Gene, Subcellular Localization and the Preparation of Transgenic Mice

 ZHU  Cai-Ye, WEI  Guang-Hui, LI  Wei, WANG  Dan, ZHENG  Meng-Meng, LIU  Zhi-Yong, ZHANG  Ya-Ni, LI  Bi-Chun   

  1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu
  • Received:2013-02-02 Online:2013-09-01 Published:2013-05-28

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Abstract: 【Objective】The aim of this paper was to clone the stearoyl-CoA desaturase 1(SCD1) gene from Xuhuai goat, and analyze it’s sub-cellular localization through enhanced green fluorescent (EGFP) fusion protein. Study the gene expression in heterologous animals, explore the possibility of preparing dissimilar transgenic animals and verify whether the foreign gene can stably inherited. 【Method】cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR), it’s biological information characteristics was analyzed by online software, and fusion expression vector named pEGFP-C1-SCD1 was constructed successfully. Then NIH-3T3 cells were transfected with pEGFP-C1-SCD1 through lipofectamine TMLTX&PLUS(LTX). At 48 h post-transfection, the transfected cells were observed under fluorescence inverted microscope. Then the mRNA expression level of SCD1 was detected by RT-PCR. The transgenic mice was produced by testicular injection, and the target gene expression in the F1 and F2 generations of individual was detected at DNA and protein levels. 【Result】 The results showed that the coding sequence (CDS) length of Xuhuai goat SCD1 gene was 1 074 bp, encoding 357 amino acids with GenBank accession number (JX854036). The SCD1 cDNA coding sequence was compared with the corresponding region of Human, Rattus norvegicus, Mus, Ovis aries, Bos taurus, Sus scrofa, respectively, the similarity was 86%, 83%, 82%, 98%, 95%, and 90%, respectively, and amino acid sequence homology was 84%, 79%, 79%, 96%, 92%, and 95%, respectively. pEGFP-SCD1 was successfully constructed. The RT-PCR results showed the SCD1 mRNA expressed successfully in vitro. The SCD1 protein was localized in the cytoplasm which was in line with the result of online prediction. The gene can also be expressed in mice transiently and persistencely after intravenous and testicular injection and the gene can be genetically stable. 【Conclusion】The SCD1 gene cDNA of Xuhuai goat was cloned successfully and in the single-cell level can express in the cytoplasm, also can express in mice.

Key words: Xuhuai goat , stearoyl-CoA desaturase , eukaryotic expression , NIH-3T3 cells , GFP , transgenic mice

[1]Los D A, Murata N. Structure and expression of fatty acid desaturases. Biochim Biophys Acta Lipids Lipid Metabology, 1998, 1394: 3-15.

[2]Thiede M A, Ozols J, Strittmatter P. Construction and sequence of cDNA for rat-liver stearyl Coenzyme-A desaturase. Journal of Biological Chemistry, 1986, 261: 3230-3235.

[3]Sam Path H, Miyazaki M, Dobrzyn A, Ntambi J. Stearo y-l CoA desaturase-1 mediates the pro-lipog eniceffects of dietary saturated fat. Journal of Biological Chemistry, 2007, 282(4): 2483-2493.

[4]Mele M, Cont e G, Castiglioni B, Chessa S, Macciotta N P P, Serra A, Buccioni A, Pagnacco G, Secchiari P. Stearoyl-coenzyme A desaturase gene polymorphism and milk fatty acid composition in Italian Holsteins. Journal of Dairy Science, 2007, 90(9): 4458-4465.

[5]陶璇, 张健, 魏学良, 牛SCD基因研究进展. 专论与综述, 2009, 29(4): 57-59.

Tao X, Zhang J, Wei X L. Bovine SCD gene research progress. Monographs and Review, 2009, 29(4): 57-59. (in Chinese)

[6]Chen Z Q, Sun J J, Li Z J, Lan X Y, Zhang C L, Qu Y J, Liu Y L, Fang X T, Lei C Z, Chen H. Novel SNPs in the caprine stearoyl-CoA desaturase (SCD) and decorin (DCN) genes that are associated with growth traits in Chinese goat breeds. Molecular Biology Reports， 2011, 38: 3121-3127.

[7]Kim J H, Jung-Ha H S, Lee H T, Chung K S. Development of a positive method for male stem cell mediated gene transfer in mouse and pig. Molecular Reproduction and Development, 1997, 46(4): 515-526.

[8]Farre L, Rigau T, Mogas T, García-Rocha M, Canal M, Gomez-Foix A M, Rodriguez-Gil J E. Adenovirus-mediated introduction of DNA into pig sperm and offspring. Molecular Reproduction and Development, 1999, 53(2): 149-158. 

[9]Giuili G, Tomljenovic A, Labrecque N, Oulad-Abdelghani M, Rassoulzadegan M, Cuzin F. Murine spermatogonial stem cells: targeted transgene expression and purification in an active state. EMBO Reports, 2002, 3(8): 753-759.

[10]沈新明, 乔贵林, 张玲, 江培洲, 黄华, 姚开泰. 用曲细精管微注射法建立绿色荧光蛋白转基因小鼠. 第一军医大学学报, 2002, 22(3): 250-253.

Shen X M, Qiao G L, Zhang L, Jiang P Z, Huang H, Yao K T. Construction of transgenic mice carrying enhanced green fluorescent protein gene by seminiferous tubule microinjection. Journal of First Military Medical University, 2002, 22(3): 250-253. (in Chinese)

[11]肖红卫, 郑新民, 陈思怀, 乔宪凤, 谷习文, 田永祥, 张涌, 魏庆信. 精子介导生产转hCD59基因猪. 华中农业大学学报, 2006, 25(2): 170-173.

Xiao H W, Zheng X M, Chen S H, Qiao X F. Gu X W, Tian Y X, Zhang Y, Wei Q X. Sperm-mediated gene transfer to produce hCD59 transgenic porcine. Journal of Huazhong Agricultural University, 2006, 25(2): 170-173. (in Chinese)

[12]Ward R J, Travers M T, Richards S E, Vernon R G, Salter A M, Buttery P J, Barber M C. Stearoyl-CoA desaturase mRNA is transcribed from a single gene in the ovine genome. Biochimica et Biophysica Acta, 1998, 1391: 145-156.

[13]刘自增, 阎萍. 天祝白牦牛 Lept in 和SCD1基因单核苷酸多态性与肌肉脂肪酸含量的关联性分析. 中国畜牧兽医, 2011, 38(4): 154-158.

Liu Z Z, Yan P. Association analyses of SNPs in the Lpetin and SCD1 gene on the fatty acid content of intramuscular fat in Tianzhu White Yak. China Animal Husbandry & Veterinary Medicine, 2011, 38(4): 154-158. (in Chinese)

[14]Lavitrano M, Camaioni A, Fazio V M, Dolci S, Farace M G. Spadafora C(1989) sperm cells as vectors for introducing foreign DNA into eggs: genetic transformation of mice. Cell, 57: 717-723.

[15]Jiang J J, Yamato Eiji, Miyazaki Jun-ichi. Intravenous delivery of naked plasmid DNA for in vivo cytokine expression. Biochemical and Biophysical Research Communications, 2001, 289(5): 1088-1092.

[16]Bachiller D, Schellander K, Peli J. Rüther U. Liposome-mediated DNA uptake by spem cells. Molecular Reproduction and Development, 1991, 30: 194-200.

[17]Lavitrano M, Forni M, Bacci M L, Di S C, Varzi V, Wang H, Seren E. Sperm mediated gene transfer in pig: Selection of donor boars and optimization of DNA uptake. Molecular Reproduction and Development, 2003, 64: 284-291.

[18]Nakanishi A, Iritani A. Gene transfer in the chicken by sperm- mediated methods. Molecular Reproduction and Development, 1993, 36: 258-261.

[19]Xie C, Yu M J, Hui N, Wang L B, Li W J, Zhao Y J. Testis injection of exogenous DNA results in expression in mouse sperm. Chinese Journal of Zoology, 2010, 2: 151-157.

[20]Hammer R E. Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth. Nature, 1985, 316: 680-683.

[21]常慧云, 谢庆阁, 陶涛, 冯书堂, 侯顺利. 外源基因导入猪受精卵的研究. 畜牧与兽医, 1993(5): 196-197.

Chang H Y, Xie Q G, Tao T, Feng S T, Hou S L. The study on foreign gene import pig zygotes. Acta Veterinaria et Zootechnica, 1993(5): 196-197. (in Chinese)

[22]Houdebine L M. The methods to generate transgenic animals and to control transgene expression. Biotechnology, 2002, 98(2/3): 145-160.

[23]Aigner A, Jäger M, Pasternack R, Weber P, Wienke D, Wolf S. Purification and characterization of cys-teine-S-conjugate N-acetyltransferase from pig kidney. Biochemical Journal, 1996, 317(pt1): 213-218.
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