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| Construction of Controllable Mus Musculus Growth Hormone(GH) shRNA Expression Vector and Its Gene Silencing Efficiency |
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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.
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Received: 23 April 2015
Published: 11 October 2015
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