[Construction of eukaryotic expression vector of mTSARG3 and establishment of its stable transfected cell line].
OBJECTIVE
To study the function of mTSARG3, the eukaryotic expression vector of mouse mTSARG3 and its stable transfected GC-1 cell line was established.


METHODS
The open reading frame (ORF) of mTSARG3 was amplified from mouse testis cDNA library by PCR. The products were cloned into pGEM-T Easy vectors and sequenced. Then the recombinant plasmid was digested by Not I and Hind III. The target cDNA fragment was subcloned into pcDNA3.1 Hygro(-), a eukaryotic expression vector. The recombined plasmid pcDNA3.1 Hygro(-)/mTSARG3 was identified and transfected into GC-1 spg cell. After screening culture by hygromycin, stable transfected GC-1 cell line was established, and the expression of mTSARG3 was identified by RT-PCR and Western blot. The flow cytometry was adopted to observe the influence on cell cycle, and the apoptosis of GC-1 cells transfected with pcDNA3.1 Hygro(-)/mTSARG3 and pcDNA3.1 Hygro(-), respectively.


RESULTS
The eukaryotic expression plasmid of pcDNA3.1 Hygro(-)/mTSARG3 was successfully constructed and stable transfected GC-1 cell line was established. RT-PCR and Western blot result revealed that mTSARG3 was expressed successfully in GC-1 cells. The flow cytometer shows that the cell transfected with pcDNA3.1 Hygro(-)/mTSARG3 could accelerate the growth and reduce the apoptosis of GC-1 cells.


CONCLUSIONS
The recombinant eukaryotic expression vector of pcDNA3.1 Hygro(-)/mTSARG3 has been constructed successfully and stably expressed in GC-1 cell line. The establishment of mTSARG3 stable transfected GC-1 spg cell line will provide a foundation for further studies on the function of mTSARG3 in vitro.
Liu
Gang
G
Institute of Reproduction and Stem Cell Engineering, Central South University, Changsha 410078, China.
Lu
Guang-Xiu
GX
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D002453:Cell Cycle; D002460:Cell Line; D045744:Cell Line, Tumor / Q000473:pathology; D018076:DNA, Complementary / Q000235:genetics; D056890:Eukaryota / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression / Q000502:physiology*; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D014162:Transfection / Q000379:methods*; D018832:Molecular Chaperones; D051017:Apoptosis Regulatory Proteins
D018076:DNA, Complementary; D006360:Heat-Shock Proteins; C531170:DNAJB13 protein, mouse; D018832:Molecular Chaperones; D051017:Apoptosis Regulatory Proteins