Journal of Zhejiang University SCIENCE
(ISSN 1009-3095, Monthly)

2005   Vol. 6B   No. 7   p.673-677

Construction and identification of Fas-targeting siRNA-expressing plasmid

LIU Su-hu¹, ZHANG Wang-gang^†1, ZHANG Mei², ZHU Qing¹, TIAN Wei¹

(¹Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, Department of Clinical Hematology, Second Hospital, Xi’an Jiaotong University, Xi’an 710004, China)
(²Department of Pathology, Second Hospital, Xi’an Jiaotong University, Xi’an 710004, China)
^†E-mail: zhangwanggang2003@yahoo.com
Received Sept. 22, 2004; revision accepted Dec. 24, 2004

Abstract: 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. Conclusion: 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 words: RNA interference, Fas protein, Plasmid, Vector construction
doi:10.1631/jzus.2005.B0673             CLC number: R349.8