[Construction of eukaryotic expression plasmid for a novel zinc finger protein gene Zfp637 and its effect on breast carcinoma cell EMT6].
OBJECTIVE
To construct the eukaryotic expression plasmid and establish stably transfected cell line, which contained the code gene of zinc finger protein637 (Zfp637), and to observe the effect of Zfp637 gene to the proliferation of tumor cells.


METHODS
The Zfp637 DNA was amplified from the template of normal spleen tissue cDNA and cloned into the eukaryotic expression vector pEGFP-C3. The recombinant plasmid, named as pEGFP-Zfp637, was determined by restriction enzyme and sequencing analyses. Next the pEGFP-Zfp637 recombinant plasmid was transferred into mouse breast carcinoma EMT6 cells with lipofectamine 2000, and the stably transfected cells were selected by G418 (named Zfp637-EMT6). The growth condition of cells was observed, and subcellular localization of Zfp637 gene was located by fluorescence microscope at the same time. The Zfp637 mRNA expression in the transfected cells was detected by RT-PCR, and the proliferation of such cells was measured by MTT.


RESULTS
The analysis confirmed that the recombinant pEGFP-Zfp637 contained the Zfp637 full-length cDNA. The Zfp637 mRNA was over-expressed stably in Zfp637-EMT6. The growth of Zfp637-EMT6 was increased obviously when compared with the negative control group and blank group.


CONCLUSIONS
The recombinant pEGFP-Zfp637 has been constructed successfully, and the expression of the Zfp637 gene can promote the proliferation of cells. This recombinant eukaryotic expression plasmid can be used in advanced studies in the biological effects of Zfp637 and anti-tumor gene therapy.
Li
Kai
K
Geratology Laboratory, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Ren
Jing-Jing
JJ
Zhang
Jie
J
Liu
Kai
K
Qi
Yan-Yu
YY
Wang
Xiu-Jie
XJ
Xiao
Heng-Yi
HY
Lin
Ping
P
D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D015316:Genetic Therapy; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection; D016335:Zinc Fingers / Q000235:genetics*
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins