[Construction of p-hfgl2shRNA and its effect on hfgl2 expression in vitro].

Abstract

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%.

CONCLUSION:

The study demonstrated that p-hfgl2shRNA successfully interfered with hfgl2 expression in vitro. This provides a foundation to further explore its application in vivo.

• Authors: Dong Xi, Li Li, Sui Gao
• Journal: Zhonghua gan zang bing za zhi = Zhonghua ganzangbing zazhi = Chinese journal of hepatology (Zhonghua Gan Zang Bing Za Zhi) Vol. 16 Issue 12 Pg. 930-4 (Dec 2008) ISSN: 1007-3418 [Print] China
• PMID: 19105940 (Publication Type: English Abstract, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• FGL2 protein, human
• RNA, Small Interfering
• Fibrinogen

Topics

• Animals
• CHO Cells
• Cricetinae
• Cricetulus
• Fibrinogen (genetics)
• Plasmids
• RNA Interference
• RNA, Small Interfering (genetics)