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[Construction of eukaryotic expression vector of mTSARG3 and establishment of its stable transfected cell line]
1Institute of Reproduction and Stem Cell Engineering, Central South University, Changsha 410078, China.
Aim:
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.
Conclusion:
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.
