[Establishment of breast cancer MDA-MB-231 cell line stably over-expressing human TOX high mobility group box family member 3].
OBJECTIVE
To construct the lentiviral expression vector of human TOX high mobility group box family member 3 (TOX3) gene and the MDA-MB-231 cell line which stably over-expresses TOX3 gene.


METHODS
TOX3 gene was synthesized by the gene synthesis method and amplified by PCR, and then cloned into pLVEF-1a/GFP-Puro vector to construct pLVEF-1a/GFP-Puro-TOX3 lentiviral vector. After restriction enzyme analysis and sequence identification, the lentiviral vector was packaged and the titer was detected. The human breast cancer MDA-MB-231 cells were transfected with the recombinant lentiviral vector and cultured selectively by puromycin to acquire stably transfected cells. MDA-MB-231 cells which expressed GFP were observed by fluorescence microcopy. And the expression levels of TOX3 mRNA and protein in transfected MDA-MB-231 cells were detected by real-time quantitative PCR(qRT-PCR) and Western blotting, respectively.


RESULTS
Restriction enzyme digestion and sequence analysis demonstrated that the lentiviral expression vectors of pLVEF-1a/GFP-Puro and pLVEF-1a/GFP-Puro-TOX3 were successfully constructed, and the viral titers were respectively 2×10(8) TU/mL and 1×10(8) TU/mL after lentiviral packaging. And after being transfected, more than 95% cells expressed GFP under a fluorescence microscope. The results of qRT-PCR and Western blotting showed that, when compared with the MDA-MB-231-NC negative control group, the expression of TOX3 mRNA and protein significantly increased in the MDA-MB-231-TOX3 group.


CONCLUSIONS
The study successfully constructed lentiviral expression vector of TOX3 gene and obtained MDA-MB-231 cell line stably over-expressing TOX3 gene by transfection with the recombinant vector.
Basic Medical College, Heilongjiang University of Chinese Medicine, Harbin 150040, China.
Cuicui
C
Han
Medical Science Research Center, Qiqihar Medical University, Qiqihar 161006, China.
Liling
L
Yue
Basic Medical College, Heilongjiang University of Chinese Medicine, Harbin 150040, China.
Ying
Y
Yang
Medical Science Research Center, Qiqihar Medical University, Qiqihar 161006, China.
Baiyu
B
Jian
Medical Science Research Center, Qiqihar Medical University, Qiqihar 161006, China.
Liwei
L
Ma
Basic Medical College, Heilongjiang University of Chinese Medicine, Harbin 150040, China.
Jicheng
J
Liu
D051017:Apoptosis Regulatory Proteins; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D005260:Female; D006609:High Mobility Group Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D010957:Plasmids; D011980:Receptors, Progesterone / Q000235:genetics*; D015534:Trans-Activators; D014162:Transfection
D051017:Apoptosis Regulatory Proteins; D006609:High Mobility Group Proteins; D011980:Receptors, Progesterone; C521752:TOX3 protein, human; D015534:Trans-Activators