Abstract：Growth hormone (GH) screened by the pituitary, is a polypeptide hormone which promotes the growth of the animal body, muscle development, and regulates metabolism and other important physiological functions. In this study, a pair of Mus musculus GH (mGH) short hairpin RNA(shRNA) was designed based on the 340bp-abasic site of mGH mRNA sequence, and the mGH shRNA sequence was linked to a integrated tetracycline induced expression vector with sequences syntheses method, the recombinant vector named as pSingle-tTS-mGH shRNA-RFP(red fluorescent protein). The recombinant vector was transfected to mice pituitary tumor cell lines AtT-20 and screened with G418 two weeks to enrich cell clones which integration of exogenous genes. The RFP expression was observed under a fluorescent microscope and the GH expression levels were detected with qRT-PCR and Western blot methods. The results showed, little RFP signal could be observed in control cell groups (cell groups transfected with pSingle-tTS-RFP plasmid) and experimental cell groups (cell groups transfected with pSingle-tTS-mGH shRNA-RFP plasmid) induced with doxycyclin (DOX), the RFP signal was obviously enhance in the above 2 cell groups. The mGH mRNA in control cell groups, control cell groups adding DOX, experimental groups and experimental groups induced with DOX was 1±0.17, 1.03±0.12, 0.758±0.19 and 0.204±0.07, respectively. Compared with the other 3 groups, the GH mRNA expression in experimental groups induced with DOX (working concentration, 600 ng/μL) difference between them was extremely obvious(P＜0.01), whereas the difference among the other 3 groups was not obvious (P＞0.05). The average GH protein expression level in control cell groups, control cell groups adding DOX, experimental groups and experimental groups induced with DOX was 1, 1.07, 0.88 and 0.32, respectively. An effective mGH shRNA sequence was designed in this study and a controllable mGH shRNA overexpression vector was constructed successfully and it showed well-controlled gene expression efficiency. The GH shRNA controllable expression system prepared in this study is a good tool to study the GH autocrine / paracrine interactions, function on body development and related disease through increase and decrease the GH expression in vitro and in vivo.
[1]Bartke A, Chandrashekar V, Turyn D, et al.Effects of growth hormone overexpression and growth hormone resistance on neuroendocrine and reproductive functions in transgenic and knock-out mice[J].Proceedings of the Society for Experimental Biology and Medicine, 1999, 222(2):113-123
[2]Cullen B R .Induction of stable RNA interference in mammalian cells[J].Gene Therapy, 2006, 13(6):503-508
[3]Frohman L A, Downs T R, Chomczynski P, et al.Regulation of growth hormone-releasing hormone gene expression and biosynthesis[J].Yale Journal of Biology and Medicine, 1989, 62(5):427-433
[4]Gossen M and Bujard H.Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. [J].Proceedings of the National Academy of Sciences, 1992, 89(12):5547-5551
[5]Ju H, Bai L, Yang Y, et al. Effects of Introns on the Regulation of Porcine Growth Hormone Expression. [J].Journal of Animal and Veterinary Advances, 2013, 12(5):892-896
[6]Ju H, Zhang J, Bai L, et al.The transgenic cloned pig population with integrated and controllable GH expression that has higher feed efficiency and meat production. [J].Scientific Report, 2015, 1(1):accept-1
[7]Kleinhammer A, Wurst W, Kuhn R.Target validation in mice by constitutive and conditional RNAi[J].Methods in molecular biology, 2013, 986:307-323
[8]Lim S V, Marenzana M, Hopkinson M, et al.Excessive Growth Hormone Expression in Male GH Transgenic Mice Adversely Alters Bone Architecture and Mechanical Strength. [J].Endocrinology, 2015, 156(4):1362-1371
[9]Palmiter R D, Norstedt G, Gelinas R E, et al.Metallothionein-human GH fusion genes stimulate growth of mice[J].Science, 1983, 222(4625):809-814
[10]Pursel V G, Bolt D J, Miller K F, et al.Expression and performance in transgenic pigs[J].Journal of Reproduction and Infertility, 1990, 40:235-245
[11]Pursel V G, Pinkert C A, Miller K F, et al.Genetic engineering of livestock. [J].Science, 1989, 244(4910):1281-1288
[12]Sigl R, Ploner C, Shivalingaiah G, et al.Development of a multipurpose GATEWAY-based lentiviral tetracycline-regulated conditional RNAi system (GLTR). [J].PLoS One, 2014, 9(5):e97764-
[13]Wieghart M, Hoover J L, McGrane M M, et al.Production of transgenic pigs harbouring a rat phosphoenolpyruvate carboxykinase-bovine growth hormone fusion gene. [J].Journal of Reproduction and Infertility, 1990, 41:89-96
[14]Xue Z, Wu M, Wen K, et al.CRISPR/Cas9 mediates efficient conditional mutagenesis in Drosophila[J].G3 (Bethesda), 2014, 4(11):2167-2173
[15]白立景, 周荣, 敖红, 等.诱导表达猪生长激素pGH载体系统构建及其在PK15细胞中表达验证[J].农业生物技术学报, 2013, 21(4):483-488
[16]鞠辉明, 白立景, 姜星, 等.整合型诱导表达猪生长激素(pGH)载体的构建及表达研究[J].畜牧兽医学报, 2013, 44(7):1008-1013
