Subtype- and species-specific knockdown of PKC using short interfering RNA.
RNA interference (RNAi), the targeted mRNA degradation induced by double-stranded RNA (dsRNA), is a powerful tool for analyzing gene function in many organisms. Recently, it has been shown that RNAi is also applicable to cultured mammalian cells by using short interfering RNA (siRNA) [Nature 411 (2001) 494]. To examine whether this siRNA method is useful for analyzing the subtype-specific functions of protein kinase C (PKC), we first prepared siRNAs which target human alphaPKC and human deltaPKC and applied them into mammalian cells to suppress the expression of endogenous alphaPKC and deltaPKC, respectively. Each siRNA for alpha or deltaPKC specifically suppressed the endogenous expression of corresponding PKC subtype in human-derived cell lines such as HEK-293 and HeLa cells, but not in cells derived from rat species. The suppression level of deltaPKC reached maximum 48-72h after the transfection of siRNA. In addition, the siRNA targeting rat deltaPKC suppressed endogenous and exogenous rat deltaPKCs but not human deltaPKC, suggesting that siRNAs targeting PKCs effectively knocked down endogenous/exogenous PKCs in mammalian cells, in subtype- and species-specific manner. Furthermore, we also developed the method to discriminate the siRNA-transfected cells using the antibody recognizing thymine dimer. Our present results strongly suggest that siRNA method enable us to examine the subtype-specific function of PKC, not only by knockdown of the endogenous target PKC subtype, but also by subsequent compensation with the exogenous corresponding wild/mutant PKC derived from other species.
Irie
Naoki
N
Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Japan.
Sakai
Norio
N
Ueyama
Takehiko
T
Kajimoto
Taketoshi
T
Shirai
Yasuhito
Y
Saito
Naoaki
N
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011493:Protein Kinase C / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014162:Transfection
D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C