[Construction of p-hfgl2shRNA and its effect on hfgl2 expression in vitro].
OBJECTIVE
Our previous studies have shown that an anti-sense plasmid to mouse fibrinogen like protein 2 (mfgl2) significantly reduced mfgl2 expression in vivo, markedly ameliorated inflammatory infiltration, fibrin deposition and hepatocyte necrosis, prolonged the survival time period and elevated the survival rate in Balb/cJ mice with murine hepatitis virus type 3 (MHV-3) induced fulminant hepatitis. This study was designed to explore the opportunity of RNA interference technique in the inhibitory application of hfgl2 expression, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/xeno transplantation and fetal loss syndrome.


METHODS
A plasmid named p-hfgl2shRNA complimentary to the sequence responsible for hfgl2 was constructed; meanwhile irrelevant shRNA plasmid with a random combination of the p-hfgl2shRNA sequence was used as a control. A plasmid named pEGFP-hfgl2 expressing hfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-hfgl2shRNA on the hfgl2 expression. By cotransfection of the constructed p-hfgl2shRNA and pEGFP-hfgl2 or pcDNA3.1-hfgl2 expression plasmids into CHO cells, the inhibition of hfgl2 expression by hfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, real time PCR and immunohistochemistry staining.


RESULTS
The experiments showed a significant inhibitory effect of p-hfgl2shRNA on hfgl2 expression at 48 h post-cotransfection by observation of green fluorescent cells, FACS, real time PCR and immunohistochemistry staining, with the inhibitory efficiency reaching as high as 85.5%.


CONCLUSIONS
The study demonstrated that p-hfgl2shRNA successfully interfered with hfgl2 expression in vitro. This provides a foundation to further explore its application in vivo.
Xi
Dong
D
Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Li
Li
L
Gao
Sui
S
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005340:Fibrinogen / Q000235:genetics*; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
C095202:FGL2 protein, human; D034741:RNA, Small Interfering; D005340:Fibrinogen