PiggyBac转座子介导的转FGF5s基因绒山羊胎儿成纤维细胞的制备

doi:10.11843/j.issn.0366-6964.2013.12.010

Abstract

Abstract:

The objective of the present study is to establish the Cashmere goat fetal fibroblasts cell line that stably expresses Cashmere goat FGF5s. By using RT-PCR technology, the FGF5s gene of Cashmere goat was first cloned from total RNAs which extracted from Cashmere goat skin, and then inserted into the eukaryotic expression vector PB-CMV-Puro-EGFP based on PiggyBac transposon. PB-EGFP-FGF5s plasmid and PB transposase plasmid were used to co-transfect the Cashmere goat fetal fibroblasts by Lipofectamine^TM2000. A stable fibroblast line which expressed green fluorescence was obtained using Puromycin screening. The integration of exogenous DNA and the exogenous mRNA expression of FGF5s were identified through PCR and RT-PCR, respectively. The complete CDS area of FGF5s was cloned and then the PB-EGFP-FGF5s plasmid was constructed successfully. The present results of the transgenic cell clones identification illustrated that the exogenous FGF5s gene was integrated into the Cashmere goat genome. Moreover, mRNA of FGF5s was detected to express in fibroblast cell line according to the RT-PCR data. Collectively, this study provides an effective approach for preparing transgenic cells of Cashmere goat, and a basis for nuclear transplantation in the future.

CLC Number:

S827
S814.8

HE Xiao-lin, YUAN Chao, QU Lei, CHEN Yu-lin. Cloning of FGF5s Gene from Cashmere Goat and Its Stable Transfection of Caprine Fetal Fibroblasts Based on PiggyBac Transposon[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(12): 1926-1931.

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Cloning of FGF5s Gene from Cashmere Goat and Its Stable Transfection of Caprine Fetal Fibroblasts Based on PiggyBac Transposon

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