Lentivirus-delivered stable gene silencing by RNAi in primary cells

Sheila A. Stewart, Derek M. Dykxhoorn, Deborah Palliser, Hana Mizuno, Evan Y. Yu, Dong Sung An, David M. Sabatini, Irvin S Y Chen, William C. Hahn, Phillip A. Sharp, Robert A. Weinberg, Carl D. Novina

Research output: Contribution to journalArticle

705 Citations (Scopus)

Abstract

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

Original languageEnglish (US)
Pages (from-to)493-501
Number of pages9
JournalRNA
Volume9
Issue number4
DOIs
StatePublished - Apr 1 2003
Externally publishedYes

Fingerprint

Lentivirus
Gene Silencing
RNA Interference
Moloney murine leukemia virus
Genes
Genome
Reverse Genetics
Transformed Cell Line
Caenorhabditis elegans
Green Fluorescent Proteins
Tumor Suppressor Genes
Dendritic Cells
Drosophila
Transfection
Saccharomyces cerevisiae
Fibroblasts
RNA
Phenotype

Keywords

  • Dendritic cells
  • Hairpin RNA
  • Knockdown
  • Retrovirus
  • SiRNA

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

Cite this

Stewart, S. A., Dykxhoorn, D. M., Palliser, D., Mizuno, H., Yu, E. Y., An, D. S., ... Novina, C. D. (2003). Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA, 9(4), 493-501. https://doi.org/10.1261/rna.2192803

Lentivirus-delivered stable gene silencing by RNAi in primary cells. / Stewart, Sheila A.; Dykxhoorn, Derek M.; Palliser, Deborah; Mizuno, Hana; Yu, Evan Y.; An, Dong Sung; Sabatini, David M.; Chen, Irvin S Y; Hahn, William C.; Sharp, Phillip A.; Weinberg, Robert A.; Novina, Carl D.

In: RNA, Vol. 9, No. 4, 01.04.2003, p. 493-501.

Research output: Contribution to journalArticle

Stewart, SA, Dykxhoorn, DM, Palliser, D, Mizuno, H, Yu, EY, An, DS, Sabatini, DM, Chen, ISY, Hahn, WC, Sharp, PA, Weinberg, RA & Novina, CD 2003, 'Lentivirus-delivered stable gene silencing by RNAi in primary cells', RNA, vol. 9, no. 4, pp. 493-501. https://doi.org/10.1261/rna.2192803
Stewart SA, Dykxhoorn DM, Palliser D, Mizuno H, Yu EY, An DS et al. Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA. 2003 Apr 1;9(4):493-501. https://doi.org/10.1261/rna.2192803
Stewart, Sheila A. ; Dykxhoorn, Derek M. ; Palliser, Deborah ; Mizuno, Hana ; Yu, Evan Y. ; An, Dong Sung ; Sabatini, David M. ; Chen, Irvin S Y ; Hahn, William C. ; Sharp, Phillip A. ; Weinberg, Robert A. ; Novina, Carl D. / Lentivirus-delivered stable gene silencing by RNAi in primary cells. In: RNA. 2003 ; Vol. 9, No. 4. pp. 493-501.
@article{403be440c37c4cd5b3787460208dba98,
title = "Lentivirus-delivered stable gene silencing by RNAi in primary cells",
abstract = "Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.",
keywords = "Dendritic cells, Hairpin RNA, Knockdown, Retrovirus, SiRNA",
author = "Stewart, {Sheila A.} and Dykxhoorn, {Derek M.} and Deborah Palliser and Hana Mizuno and Yu, {Evan Y.} and An, {Dong Sung} and Sabatini, {David M.} and Chen, {Irvin S Y} and Hahn, {William C.} and Sharp, {Phillip A.} and Weinberg, {Robert A.} and Novina, {Carl D.}",
year = "2003",
month = "4",
day = "1",
doi = "10.1261/rna.2192803",
language = "English (US)",
volume = "9",
pages = "493--501",
journal = "RNA",
issn = "1355-8382",
publisher = "Cold Spring Harbor Laboratory Press",
number = "4",

}

TY - JOUR

T1 - Lentivirus-delivered stable gene silencing by RNAi in primary cells

AU - Stewart, Sheila A.

AU - Dykxhoorn, Derek M.

AU - Palliser, Deborah

AU - Mizuno, Hana

AU - Yu, Evan Y.

AU - An, Dong Sung

AU - Sabatini, David M.

AU - Chen, Irvin S Y

AU - Hahn, William C.

AU - Sharp, Phillip A.

AU - Weinberg, Robert A.

AU - Novina, Carl D.

PY - 2003/4/1

Y1 - 2003/4/1

N2 - Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

AB - Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.

KW - Dendritic cells

KW - Hairpin RNA

KW - Knockdown

KW - Retrovirus

KW - SiRNA

UR - http://www.scopus.com/inward/record.url?scp=0344375083&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0344375083&partnerID=8YFLogxK

U2 - 10.1261/rna.2192803

DO - 10.1261/rna.2192803

M3 - Article

VL - 9

SP - 493

EP - 501

JO - RNA

JF - RNA

SN - 1355-8382

IS - 4

ER -