[Construction of PCI-neo mammalian expression vector system containing murine 4-1BBL gene and its stable expression in hepatocellular carcinoma cell line].
OBJECTIVE
To study the construction of the PCI-neo mammalian expression vector system containing murine 4-1BBL gene and its stable and effective expression in rat hepatocellular carcinoma cell line CBRH7919.


METHODS
The murine full-length 4-1BBL cDNA was obtained and subcloned into the PCI-neo mammalian expression vector. The recombinant named as PCI-neo-4-1BBL was identificated by restriction enzyme digestion and sequencing. Subsequently, PCI-neo-4-1BBL was transfected into CBRH7919 with lipofectamine reagent, then G418-resistance clone was acquired and named as PCI-neo-4-1BBL-CBRH7919+. The stable expression of 4-1BBL mRNA in PCI-neo-4-1BBL-CBRH7919+ was detected by RT-PCR.


RESULTS
The fragment of 980 bp (4-1BBL) and 5.4 kb (PCI-neo) was shown after PCI-neo-4-1BBL had been digested by EcoR I and Not I and agarose gel electrophoresis. The DNA sequencing of 4-1BBL, proved to be identical to the data of 4-1BBL in Genebank, showed stable and effective expression in PCI-neo-4-1BBL-CBRH7919+.


CONCLUSIONS
The PCI-neo mammalian expression vector system containing murine 4-1BBL has been constructed successfully, which shows stable and effective expression in CBRH7919.
Li
Guo-qiang
GQ
Department of Liver Surgery, Jiangsu Liver Transplantation Center, First Hospital, Nanjing Medical University, Nanjing 210029, China.
Wang
Xue-hao
XH
Yin
Jie
J
Yu
Yue
Y
D053330:4-1BB Ligand; D000818:Animals; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D008114:Liver Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D011995:Recombination, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics*
D053330:4-1BB Ligand; D018076:DNA, Complementary; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha