A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.
RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.
Rubinson
Douglas A
DA
Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Dillon
Christopher P
CP
Kwiatkowski
Adam V
AV
Sievers
Claudia
C
Yang
Lili
L
Kopinja
Johnny
J
Rooney
Dina L
DL
Zhang
Mingdi
M
Ihrig
Melanie M
MM
McManus
Michael T
MT
Gertler
Frank B
FB
Scott
Martin L
ML
Van Parijs
Luk
L
D000818:Animals; D001483:Base Sequence; D016827:CD8 Antigens / Q000235:genetics; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics; D034622:RNA Interference; D015375:Receptors, Interleukin-2 / Q000235:genetics; D013234:Stem Cells
D016827:CD8 Antigens; D008164:Luminescent Proteins; D015375:Receptors, Interleukin-2; D049452:Green Fluorescent Proteins; D012313:RNA