[Construction of the p-mfgl2shRNA and its effect on mfgl2 expression in vitro].
OBJECTIVE
To construct the siRNA plasmid for mfgl2 gene, which has been reported to be involved in a variety of disease developments including fulminant viral hepatitis, acute rejection of allo/zero transplantation and fetal loss syndrome, and to investigate its inhibitory effects on mfgl2 expression in vitro.


METHODS
A plasmid p-mfgl2shRNA complimentary to the sequence responsible for the functional domain of mouse fgl2 (mfgl2) was constructed. The pcDNA3.1 mfgl2 expression construct was able to show a satisfactory fgl2 protein expression. The plasmid expression pEGFP and a construct expressing irrelevant shRNA with a random combination of the p-mfgl2shRNA sequence were used as controls. A pEGFP-mfgl2 expressing mfgl2-EGFP fusion protein was also constructed for screening of the effect of p-mfgl2shRNA on the mfgl2 expression.


RESULTS
Cotransfection of p-mfgl2shRNA with pEGFP-mfgl2 decreased green fluorescent cells and the lightness of fluorescence within the cells at the 24 h, 48 h and 72 h post-transfection when compared with that in the control groups which were solely transfected with pEGFP-mfgl2. Furthermore the mfgl2 expression was significantly reduced when the pcDNA3.1 mfgl2 expression construct was cotransfected with p-mfgl2shRNA both at mRNA level by RT-PCR and protein level by RT-PCR, immunohistochemistry staining and FACS in both CHO cell and Hela cell lines.


CONCLUSIONS
The study demonstrated that the construct of p-mfgl2shRNA successfully interfered in the mfgl2 expression in vitro. It provides a basis for a further investigation of effect in vivo.
Wang
Zhi-mo
ZM
Laboratory of Infectious Immunology and Department of Infectious Diseases, Tongji Hospital of Tongji Medical College, Hua Zhong University of Science and Technology, Wuhan 430030, China.
Yan
Wei-ming
WM
Xi
Dong
D
Zhu
Chuan-long
CL
Luo
Xiao-ping
XP
Ning
Qin
Q
D000818:Animals; D005340:Fibrinogen / Q000096:biosynthesis* / Q000235:genetics*; D015870:Gene Expression; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*
C490637:Fgl2 protein, mouse; D034741:RNA, Small Interfering; D005340:Fibrinogen