[A study on construction of plasmid pcDNA3.1 (-) CMV.CD and transfection into laryngeal cancer cell Hep-2].
OBJECTIVE
To construct a recombinant plasmid containing the yeast germ suicide gene CD and explore the killing effect of CD gene on Hep-2 cells in combination with 5-FC in vitro.


METHODS
A fragment containing full-length coding region of CD was subcloned into expression plasmid pcDNA3.1 (-) CMV to construct recombinant plasmid pcDNA3.1 (-) CMV. CD identified by enzyme digestion of XhoI/HindIII, then the recombinant plasmid was conducted into the Hep-2 cell line by electroporation. Hep-2 cells stably expressing CD was obtained by 14-day positive select with 300 approximately 600 mg/L G418 and negative select with 10 mg/L 5-FC. Total RNA was extracted and the expression of the CD gene in transfected Hep-2 cells was identified by RT-PCR. MTT assay was used to observe the killing effect of 5-FC of different concentration on Hep-2 cells stably expressing CD and the controlled group was Hep-2 cells transfected by pcDNA3.1 (-) CMV.


RESULTS
A fragment of 5353bp and inserted fragment of 496 bp were got by cutting positive recombinant plasmid of pcDNA3.1 (-) CMV. CD with XhoI/HindIII. Automatic DNA sequence analysis demonstrated that the recombinant plasmid pcDNA3.1 (-) CMV. CD contained integral coding region of CD of 477bp that was totally the same with that published in GenBank. The expression of CD gene was detected by RT-PCR. The relative survival rate of Hep-2 cells stably expressing CD were separately lower than those in the control group (P < 0.05).


CONCLUSIONS
pcDNA3.1 (-) CMV. CD was successfully constructed and CD-expressing Hep-2 cells can be killed by 5-FC, so the recombinant plasmid may be a candidate vector for laryngeal cancer therapy.
Department of Otolaryngology, Xiangya Hospital, Central South University, Changsha, 410078, China. shiw888@yahoo.com.cn
Yaoyun
Y
Tang
Jianyun
J
Xiao
Suping
S
Zhao
Yong
Y
Feng
Hongru
H
Zhang
D045744:Cell Line, Tumor; D015870:Gene Expression; D041722:Genes, Transgenic, Suicide; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger; D014162:Transfection
D012333:RNA, Messenger