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Arch Med Sci. 2011 Aug;7(4):579-85. doi: 10.5114/aoms.2011.24124. Epub 2011 Sep 2.

Construction of eukaryotic expression vector of TSLC1 gene.

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1
Department of Oncology, Affiliated Hospital of Guangdong Medical College, Zhanjiang, China.

Abstract

INTRODUCTION:

To construct a eukaryotic expression vector of the tumour suppressor in lung cancer 1 (TSLC1) gene, so as to explore the mechanisms of tumour suppression of the gene theoretically.

MATERIAL AND METHODS:

The open reading frame (ORF) of TSLC1 gene was amplified with RT-PCR from normal human foreskin acrobystia, and cloned to pMD19-T simple vector (TA Clone method). The resultant plasmid was transformed into Escherichia coli JM109 for amplification. The TA Clone recombinant was digested by double restriction enzyme (Bgl II/EcoR I) and analysed with agarose gel electrophoresis. The positive one was sequenced. The inserted DNA fragment was recovered, and then it was mounted into the eukaryotic expression vector pIRES2-EGFP, transformed into E. coli JM109 for amplification. A positive recombinant plasmid named pIRES2-EGFP-TSLC1 was confirmed by Bgl II/EcoR I double-enzyme digestion analysis.

RESULTS:

RT-PCR amplified the ORF of the TSLC1 gene. It was approximately 1400 base pairs. The obtained DNA was confirmed a high degree of homology with the sequence of TSLC1 cDNA sequence (AY358334) stored at GenBank.

CONCLUSIONS:

Construction of a TSLC1 eukaryotic expression vector was successful, and it has established a solid foundation for further study.

KEYWORDS:

TSLC1 gene; construction; eukaryotic expression vector

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