Optimization of short hairpin RNA for lentiviral-mediated RNAi against WAS.
The expression of short hairpin RNAs (shRNAs) with lentiviral vectors is useful to induce stable RNA interference, particularly in hematopoietic cells. Since primary cells integrate few copies of vector, we tested if several shRNA cassette modifications could improve knock-down efficacy. Using two shRNA sequences previously shown to inhibit the human WAS gene expression, we found that neither increasing the shRNA stem length from 19-nt to 29-nt, nor modifying the loop with 4-nt, 9-nt artificial loops or with the mir30 loop improved vector-induced shRNA efficacies. This cautions against extrapolating results obtained with synthetic molecules to shRNAs that are stably expressed from viral vectors. On the other hand, the duplication of the shRNA expression cassette resulted in twice as much knock-down per copy of integrated vector. This strategy allowed a strong suppression of WASp in CD34(+) cells and will facilitate future studies on the role of WASp in human cells.
Jeanson-Leh
Laurence
L
INSERM, U790, Evry F-91002, France;Univ Paris-Sud, UMR_S790, Orsay CEDEX, F-91405 France.
Blondeau
Johanna
J
Galy
Anne
A
D015088:2',5'-Oligoadenylate Synthetase / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D009690:Nucleic Acid Conformation; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D035362:Transcription Factor TFIID / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D035362:Transcription Factor TFIID; C495767:WAS protein, human; D051316:Wiskott-Aldrich Syndrome Protein; D049452:Green Fluorescent Proteins; C475085:OAS1 protein, human; D015088:2',5'-Oligoadenylate Synthetase