Transient RNA interference in hematopoietic progenitors with functional consequences.
Short interfering (si) RNAs have now been shown to inhibit gene expression in several species, including mammals (Elbashir et al.: Nature 411:494-498, 2001; Fire et al.: Nature 391:806-811, 1998). RNA inhibition in primary cells such as stem cells would facilitate rapid gene discovery in a postgenome era. While retroviruses can deliver siRNA expression cassettes for stable expression (Barton and Medzhitov: Proc Natl Acad Sci USA 99:14943-14945, 2002; Paddison et al.: Proc Natl Acad Sci USA 99:1443-1448, 2002; Rubinson et al.: Nat Genet 33:401-406, 2003), an efficient method for direct transfer of siRNA to stem cells is still lacking. Here, we established electroporation to deliver siRNA to hematopoietic progenitors. On average, at least 80% of cells take up the RNA, and these display nearly 100% knockout of marker gene expression at both the RNA and protein level. Moreover, knockdown of the hematopoietic regulator, CD45, results in 3-fold more hematopoietic colonies in a progenitor assay. These results demonstrate that transient transfection of siRNA to primary cells can have substantial functional consequences. This technology may be applicable to a variety of primary cell types.
Oliveira
Daniela M
DM
Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas 77030, USA.
Goodell
Margaret A
MA
D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D003114:Colony-Forming Units Assay; D018274:Electroporation; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000378:metabolism*; D020868:Gene Silencing; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D019076:Transgenes
D000950:Antigens, Ly; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D017493:Leukocyte Common Antigens