[Construction of eukaryotic expression vector of human TSARG4 and establishment of its stable transfected HeLa cell line].
OBJECTIVE
To construction of eukaryotic expression vector of human TSARG4 and establishment of its stable transfected Hela cell line.


METHODS
The open reading frame (ORF) of TSARG4 was amplified from human testis by RT-PCR. The PCR products were cloned into pUCm-T vectors and sequenced. Then the cDNA fragment was subcloned into pcDNA3.1(+), a eukaryotic expression vector. The recombined plasmid pcDNA3.1(+)/TSARG4 was sequenced and transfected into Hela cell by lipofectamine 2000. After screening culture by G418, stable transfected HeLa cell line was established, and the expression of TSARG4 was identified by RT-PCR and in situ hybridization.


RESULTS
The eukaryotic expression plasmid of pcDNA3.1(+)/TSARG4 was successfully constructed and stable transfected HeLa cell line was established. RT-PCR and in situ hybridization result revealed that TSARG4 was expressed successfully in HeLa cells.


CONCLUSIONS
The recombinant eukaryotic expression vector of pcDNA3.1(+)/TSARG4 has been constructed successfully and stably expressed in HeLa cell line, providing a foundation for further studies on the function of TSARG4 in vitro.
Yang
You-bo
YB
Department of Endocrinology, Third Hospital of Central South University, Changsha 410013, China. yangyoubo@126.com
Xiang
Hong
H
Xing
Xiao-wei
XW
D002460:Cell Line; D056890:Eukaryota / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D008565:Membrane Proteins; D011506:Proteins; C477147:SUN5 protein, human