title: [Preparation and identification of recombinant adenoviruses carrying short hairpin RNA targeting parathyroid hormone related protein of goat].
abstract: Parathyroid hormone related protein (PTHrP) has important biological functions in calcium metabolism. The aim of this study was to silence the expression of PTHrP by RNA interference and recombinant adenovirus, and to provide a material to investigate the relative functions of PTHrP in goat mammary gland epithelial cell. The Block-iT shRNA interference system was used in this experiment. We designed and synthesized two pairs of complementary single-strand DNA oligonucleotides (shRNA-322/357) targeting two different sites of PTHrP mRNA. Then the oligonucleotides were inserted into shuttle vector pENTR/CMV-GFP/U6. After detection of the interference efficiency by Western blotting, we chose pENTR/CMV-GFP/U6-322 and adenovirus backbone vector pAD/PL-DEST to produce recombinant vector pAD/PL-DEST/CMV-GFP/U6-322. The first generation recombinant adenovirus particles (AD-PTHrP-322) were produced and further amplified by transfecting HEK-293 cells. The titer of the recombinant adenovirus reached 2.0 x 1(9) PFU/mL determined by TCID50 assays. The result of real-time quantitative PCR indicated that mRNA expression levels of gene were reduced 29.2%, 68.1% and 82.6% (P < 0.05), respectively, when goat mammary gland epithelial cells were infected with AD-PTHrP-322 after 24, 48 and 72 h, in which PTHrP. Western blotting also showed that the expression of PTHrP was reduced by infecting the cells with AD-PTHrP-322. AD-PTHrP-322 has been proved with significant interference effect on expression of PTHrP.
authors.affiliation: College of Animal Science and Technology, Northwest A & F University, Yangling 712100, China.
authors.forename: Ruifang
authors.initials: R
authors.lastname: Xing
authors.forename: Huiling
authors.initials: H
authors.lastname: Zheng
authors.forename: Xuemei
authors.initials: X
authors.lastname: Liu
authors.forename: Linhui
authors.initials: L
authors.lastname: Yan
authors.forename: Junhui
authors.initials: J
authors.lastname: An
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Yang
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Zhu
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006041:Goats; D057809:HEK293 Cells; D006801:Humans; D008321:Mammary Glands, Animal / Q000166:cytology; D044162:Parathyroid Hormone-Related Protein / Q000096:biosynthesis* / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
chemical_list: D044162:Parathyroid Hormone-Related Protein; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins