[Construction of epithelia membrane protein 1 gene-deficient in human fetal nucleus pulposus cell model by lentivirus -mediated RNA interference].
OBJECTIVE
To construct Epithelia Membrane Protein 1 gene-deficient in human fetal nucleus pulposus model by lentivirus-mediated RNA interference for building a platform for illustrating the biomechanisms role of EMP-1 during human intervertebral disc degeneration.


METHODS
The lentivirus vector with shRNA targeting EMP-1 mRNA was transected into 293FT cells by liposome. Then the lentivirus supernatant was obtained and used for infecting human fetal nucleus pulposus. The expression of GFP was observed under fluorescence microscope after 48 h. The viral particles were collected at 72 h after transfection. The efficacy of gene interference was tested by Western blot and Real-time RT-PCR. Analysis the results of the fluorescent microscope scenes and get the average values of EMP-1/GAPDH by detected the interference efficiency of various interference DNA sequences with western blot and semi quantitative RT-PCR methods.


RESULTS
The lentivirns with high titer were obtained and the EMP-1 gene deficient cell strains were obtained. Semi quantitative RT-PCR and Western blot proved the average values of EMP-1/GAPDH decreased from 0.46 to 0.32 and 0.5 to 0.25 (P < 0.01).


CONCLUSIONS
Lentivirus packaging technology can be mastered skillfully. EMP-1 gene-deficient cell models are successfully established.
Department of Orthopaedics, 309th Hospital of PLA, Beijing, China. huming309@hotmail.com
Ming
M
Hu
Yuan-Zheng
YZ
Ma
Da-Wei
DW
Li
Feng-Shan
FS
Huang
Da-Yu
DY
Yang
Tong-Lei
TL
Yang
Yu-Chuan
YC
Liu
D005333:Fetus; D057809:HEK293 Cells; D006801:Humans; D007403:Intervertebral Disc / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000235:genetics*; D014162:Transfection
D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; C097051:epithelial membrane protein-1