Construction of eukaryotic expression vector of hMTH1 gene antisense RNA

Jiang, G.-f.; Zhuang, Z.-x.; Liu, Q.-z.; He, Y.; Du, L.-t.

Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi 21(1): 57-60

2003


ISSN/ISBN: 1001-9391
PMID: 14761578
Accession: 048642189

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Abstract
To construct pEGFP-C1-T vector, an eukaryotic expression plasmid of hMTH1 gene antisense RNA. The conservative region of hMTH1 gene was amplified by RT-PCR after total RNA being extracted from human embryo lung fibroblast (HLF) and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the conservative region of hMTH1 gene was inserted into pEGFP-C1 vector reversedly and pEGFP-C1-T vector was constructed. The efficiency of antisense inhibition was verified by Western blotting after cell transfection. 423 bp fragment including conservative region of hMTH1 gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGFP-C1-T vector was successfully constructed by means of recombinant DNA technology. Additionally pEGFP-C1-T vector could efficiently decrease hMTH1 protein level by 46%. The efficient expression vector of hMTH1 gene antisense RNA, pEGFP-C1-T has been constructed successfully.