[Construction of FAS-targeted RNAi-retroviral vector and its identification for biological activity].
This study was aimed to construct mouse Fas-targeting si RNA-expressing recombinant retroviral vector in order to explore the therapeutic potential of Fas inhibition by siRNA in the treatment of aplastic anemia and to provide a basia for extensive development of RNA interference techninque. The U6(+) 27 promoter cassette and siFas sequence were obtained by PCR method. The U6-siFas fragment was cloned into the multiple restriction site of pLXSN-EGFP and directly downstream of EGFP gene. The resultant retroviral vector pLXSN/EGFP-siFas was packaged using PA317 cell line and tittered using NIH3T3 cell line. P815 cells were infected by the retroviral vector. EGFP expression in P815 was observed under fluorescent microscope and Fas inhibition effect was detected by immunohistochemistry. The results indicated that successfully constructed retrovirus vector pLXSN/EGFP-siFas was could not only deliver siRNA into mammalian cells efficiently and inhibit Fas expression in P815 cells, but also could express EGFP as marker and neomycine resistance gene to allow antibiotic selection. It is concluded that the successful construction of this retroviral vector would greatly facilitate the application of RNA interference and lay the foundation for therapeutic study of Fas inhibition in the treatment of aplastic anemia.
Liu
Su-Hu
SH
Department of Hematology, The Second Hospital, Xi'an JiaoTong University, Xi'an 710004, China. liusuhu1@yahoo.com
Zhang
Wang-Gang
WG
Zhang
Mei
M
Zhu
Qing
Q
Tian
Wei
W
D000741:Anemia, Aplastic / Q000235:genetics / Q000473:pathology / Q000628:therapy; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism
D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D049452:Green Fluorescent Proteins