Construction and identification of Fas-targeting siRNA-expressing plasmid.
OBJECTIVE
To study the therapeutic potential of Fas inhibition in different diseases, a Fas-targeting siRNA (small interfering)-expressing plasmid was constructed.


METHODS
The U6 promoter cassette and siFas (small interfering RNA that inhibit Fas expression) template sequence were obtained by PCR method. They were cloned into modified pcDNA3.1. The resultant plasmid pU6-siFas was transfected into P815 cells with lipofectin2000 and selected under G-418-containing culture medium. Fas inhibition in stably transfected cells was detected by immunocytochemistry.


RESULTS
The plasmid pU6-siFas efficiently reduced the expression of Fas and conferred G-418 resistance in P815 cells.


CONCLUSIONS
The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique and lay the foundation for further study of Fas inhibition in the treatment of different diseases such as aplastic anemia and acute liver failure.
Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, Department of Clinical Hematology, Second Hospital, Xi'an Jiaotong University, China.
Su-hu
SH
Liu
Wang-gang
WG
Zhang
Mei
M
Zhang
Qing
Q
Zhu
Wei
W
Tian
D000818:Animals; D045744:Cell Line, Tumor; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D034801:Mastocytoma / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D010957:Plasmids / Q000032:analysis / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection / Q000379:methods; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism*
D034741:RNA, Small Interfering; D019014:fas Receptor