Construction and Identification of a Lentiviral Vector Expressing shRNA for RNA Interference Sheep Inhibin Alpha Subunit (INHA)

doi:10.7668/hbnxb.2011.03.005

Abstract

Abstract: To construct a lentiviral vector expressing shRNA for RNA interference of sheep inhibin alpha subunit(INHA) gene and assess its gene silencing effect in 293T cell by cotransfected with an efficiently expressional INHA vector constructed simultaneously.Make preparation for producing high quality and high fecundity transgenic sheep.Design and synthesis 4 shRNA expressing sequences(shRNA1,shRNA2,shRNA3,shRNA4)and 2 control sequences(NC1,NC2)for sheep INHA gene,and then attach them to a blank plasmid vector: pGFP-v-RS,thus 4 shRNA expressing vector(that is shRNA interfering vector) pGFP-V-RS-shRNAs and 2 negative control vector pGFP-V-RS-NCs were generated;At the same time,construct an efficiently expressional vector of sheep INHA pIRES2-eGFP-INHA,then cotransfect 4 shRNA interfering vectors and 2 negative control vectors with efficiently expressional INHA vectors into 293T cell respectively and instantaneously,assess the interference effect of 4 shRNA interfering vectors contrasting with/in comparison to the 2 negative control vectors by qPCR,choose the shRNA interfering vector with the best interference effect,and construct it into corresponding lentiviral vector.The qPCR results indicate that the shRNA interference vector pGFP-V-RS-shRNA3 has the best interference effect,and the lentiviral vector constructed by it has been proved to be successfully constructed by sequencing results.Lentiviral interference vector for sheep INHA shRNA has been constructed successfully,and make the preparation for furtherly producing high quality and high fecundity transgenic sheep.

Key words: INHA, Lentivirus, shRNA, RNAi, Sheep

CLC Number:

ZHANG Rui-jie, MIAO Xiang-yang. Construction and Identification of a Lentiviral Vector Expressing shRNA for RNA Interference Sheep Inhibin Alpha Subunit (INHA)[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2011, 26(3): 21-27. doi: 10.7668/hbnxb.2011.03.005.

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References

[1] 田秀娥,孙红霞,王永军.3个绵羊群体INHA基因的遗传多态性及对产羔数的影响[J].西北农林科技大学学报:自然科学版,2010,38(1):23-29.

[2] 周文然,储明星,孙少华,等.小尾寒羊高繁殖力候选基因INHA的研究[J].农业生物技术学报,2007,15(1):32-36.

[3] 张宁,罗军.抑制素主动免疫对青年奶山羊生殖的影响[J].安徽农业科学,2007,35(26):8244-8245.

[4] Li Han,Mao D G,Zhang D K,et al.Development and evaluation of a novel DNA vaccine expressing inhibin α (1-32) fragment for improving the fertility in rats and sheep[J].Animal Reproduction Science,2007,109(2008):251-265.

[5] Li C,Zhu Y L,Xue J H,et al.Immunization against inhibin enhances both embryo quantity and quality in Holstein heifers after superovulation and insemination with sex-sorted semen[J].Theriogenology,2009,71 (6):1011-1017.

[6] Voge J L,Parker J B,Wheaton J E.Effects of immunization against alpha-inhibin using two adjuvants on daily sperm production and hormone concentrations in ram lambs[J].Domest Anim Endocrinol,2009,37 (4):206-213.

[7] Rao D D,Vorhies J S,Senzer N,et al.siRNA vs.shRNA:sinilarities and differences[J].Adv Drug Deliv Rev,2009,61(9):746-759.

[8] 周军,李建明,杨发达,等.人CDH22基因RNAi病毒载体的构建及稳定干扰CDH22基因的表达[J].南方医科大学学报,2008,28(4):589-592.

[9] Katja Sliva,Barbara S Schnierle.Selective gene silencing by viral delivery of short hairpin RNA[J].Sliva and Schnierle Virology Journal,2010,7:248

[10] Hitz C,Steuber-Buchberger P,Delic,et al.Generation of shRNA transgenic mice[J].Methods Mol Biol,2009,530:101-129.

[11] Bos T J,DeBruyneE,HeirmanC,et al.In search of the most suitable lentiviral shRNA system[J].Curr Gene Ther,2009,9 (3):192-211.

[12] Henriksen J R,Buechner J,Lokke C,et al.Inhibition of gene function in mammalian cells using short-hairpin RNA (shRNA)[J].Methods Mol Biol,2011,703:189-204.

[13] Jia F J,Huang M,Yan Y C,etal.A strategy for constructing and verifying short hairpin RNA expression vectors[J].J RNAi Gene Silencing,2007,3(1):248-253.

[14] Milazzotto M P,Goissis M D,Feitosa W B,et al.Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos[J].Zygote,2010,18 (4):339-44.

[15] Szulc J,Aebischer P.Conditional gene expression and knockdown using lentivirus vectors encoding shRNA[J].Methods Mol Biol,2008,434:291-309.

[16] Singer O,Verma I M.Applications of lentiviral vectors for shRNA delivery and transgenesis[J].Curr Gene Ther,2008,8 (6):483-488.

[17] Zhang W,Li J,Allen S M,et al.Silencing the breast cancer resistance protein expression and function in caco-2 cells using lentiviral vector-based short hairpin RNA[J].Drug Metab Dispos,2009,37(4):737-44.

[18] Anastasov N,Klier M,Koch I,et al.Efficient shRNA delivery into B and T lymphoma cells using lentiviral vector-mediated transfer[J].J Hematop,2009,2(1):9-19.

[19] Deng L,Li G,Xi L,et al.Hepatitis B virus inhibition in mice by lentiviral vector mediated short hairpin RNA[J].BMC Gastroenterol,2009,9:73.

[20] 刘畅,宋振华,秦泽莲.人periostin干扰载体的构建及其对成纤维细胞目的基因表达的影响[J].北京大学学报:医学版,2010,42(5):503-508.

[21] 王云姣,程智刚,于鹏,等.携带DREAM基因shRNA的慢病毒载体的构建及其对坐骨神经缩窄损伤大鼠的镇痛作用[J].中南大学学报:医学版,2009,34(8):723-730.

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