Optimization of short hairpin RNA for lentiviral-mediated RNAi against WAS.

Publisher: Elsevier Country of Publication: United States NLM ID: 0372516 Publication Model: Print-Electronic Cited Medium: Print ISSN: 0006-291X (Print) Linking ISSN: 0006291X NLM ISO Abbreviation: Biochem Biophys Res Commun Subsets: MEDLINE

Publication: <2002- >: San Diego, CA : Elsevier
Original Publication: New York, Academic Press.

RNA Interference*; Lentivirus/*genetics ; RNA, Small Interfering/*genetics ; Wiskott-Aldrich Syndrome Protein/*genetics; 2',5'-Oligoadenylate Synthetase/genetics ; 2',5'-Oligoadenylate Synthetase/metabolism ; Base Sequence ; Blotting, Western ; Cell Line ; Cell Line, Tumor ; Genetic Vectors/genetics ; Green Fluorescent Proteins/genetics ; Green Fluorescent Proteins/metabolism ; HCT116 Cells ; Humans ; Nucleic Acid Conformation ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Small Interfering/chemistry ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factor TFIID/genetics ; Transcription Factor TFIID/metabolism ; Transfection ; Wiskott-Aldrich Syndrome Protein/metabolism

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.

0 (RNA, Messenger)
0 (RNA, Small Interfering)
0 (Recombinant Fusion Proteins)
0 (Transcription Factor TFIID)
0 (WAS protein, human)
0 (Wiskott-Aldrich Syndrome Protein)
147336-22-9 (Green Fluorescent Proteins)
EC 2.7.7.- (OAS1 protein, human)
EC 2.7.7.84 (2',5'-Oligoadenylate Synthetase)

Date Created: 20070828 Date Completed: 20071119 Latest Revision: 20161124

20231215

10.1016/j.bbrc.2007.08.013

17719003