Construction of PCI-neo mammalian expression vector system containing murine 4-1BBL gene and its stable expression in hepatocellular carcinoma cell line.
- Author:
Guo-qiang LI
1
;
Xue-hao WANG
;
Jie YIN
;
Yue YU
Author Information
- Publication Type:Journal Article
- MeSH: 4-1BB Ligand; Animals; Cell Line, Tumor; Cloning, Molecular; DNA, Complementary; genetics; Genetic Vectors; Liver Neoplasms, Experimental; metabolism; pathology; RNA, Messenger; biosynthesis; genetics; Rats; Rats, Wistar; Recombination, Genetic; Transfection; Tumor Necrosis Factor-alpha; biosynthesis; genetics
- From: Chinese Journal of Oncology 2003;25(4):328-331
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo 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.
METHODSThe 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.
RESULTSThe 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+.
CONCLUSIONThe PCI-neo mammalian expression vector system containing murine 4-1BBL has been constructed successfully, which shows stable and effective expression in CBRH7919.
