内蒙古白绒山羊FGF5基因克隆及其靶向shRNA表达载体的构建和鉴定

doi:10.11843/j.issn.0366-6964.2014.11.008

Abstract

Abstract:

To construct the shRNA vector（pRNAT-shRNA） for Inner Mongolia Cashmere goat fibroblast growth factor 5（FGF5）.Total RNA as templates was extracted from Inner Mongolia Cashmere goat fetal fibroblasts（GFb） cells，FGF5 CDs was amplified by PCR.Three shRNA sequences targeting FGF5 were designed and linked with interference vector pRNAT-U6.1/Neo，labeled as pRNAT-shRNA1，pRNAT-shRNA2 and pRNAT-shRNA3，which were transfected into GFb cells by lipofectamine^TM2000.FGF5 mRNA expression was detected by real time PCR.The results showed that the cloned FGF5 CDs from Inner Mongolia Cashmere goat was 813 bp in length including integral ORF which encodes 270 amino acids residues.The homology of nucleotide sequence and predicted amino acid sequences were all 99% compared with ovis aries FGF5.pRNAT-shRNA was constructed successfully.Green fluorescence could be seen from transfected cells for 48 h.FGF5 mRNA expression levels were significantly lower than control group（P<0.01） in transfected pRNAT-shRNAs groups，The interference effect of pRNAT-shRNA2 group was the best in 3 groups.pRNAT-shRNA vector may provide a novel applicable strategy for the function of FGF5 in hair follicle.

Key words: Inner Mongolia Cashmere goat, fibroblast growth factor 5（FGF5）, short hairpin RNA, real time PCR, construction of vector

CLC Number:

S827.2

BAO Wen-lei，HE Qi-buri，YAO Rui-yuan，GUO Zhi-xin，BAO Chao-getu，WANG Yan-feng，WANG Zhi-gang. Cloning FGF5 from Inner Mongolia Cashmere Goat and Construction and Identification of shRNA Expression Vectors Targeting FGF5 Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(11): 1793-1798.

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Cloning FGF5 from Inner Mongolia Cashmere Goat and Construction and Identification of shRNA Expression Vectors Targeting FGF5 Gene

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