siRNA stabilization prolongs gene knockdown in primary T lymphocytes.
RNA interference (RNAi)-mediated knockdown of target gene expression represents a powerful approach for functional genomics and therapeutic applications. However, for T lymphocytes, central regulators of immunity and immunopathologies, the application of RNAi has been limited due to the lack of efficient small interfering RNA (siRNA) delivery protocols, and an inherent inefficiency of the RNAi machinery itself. Here, we use nucleofection, an optimized electroporation approach, to deliver siRNA into primary T lymphocytes with high efficiency and negligible impairment of cell function. We identify siRNA stability within the cells as the critical parameter for efficient RNAi in primary T cells. While generally short-lived and immediately lost upon T-cell activation when conventional siRNA is used, target gene knockdown becomes insensitive to cell activation and can persist for up to 2 wk in non-dividing cells with siRNA stabilized by chemical modifications. Targeting CD4 and the transcription factor GATA-3, we show that the use of stabilized siRNA is imperative for functional gene analysis during T lymphocyte activation and differentiation in vitro as well as in vivo.
Mantei
Andrej
A
Deutsches Rheuma-Forschungszentrum Berlin, Berlin, Germany.
Rutz
Sascha
S
Janke
Marko
M
Kirchhoff
Dennis
D
Jung
Ulrike
U
Patzel
Volker
V
Vogel
Uwe
U
Rudel
Thomas
T
Andreou
Ioanna
I
Weber
Martin
M
Scheffold
Alexander
A
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010047:Ovalbumin / Q000276:immunology; D034622:RNA Interference; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D034741:RNA, Small Interfering; D010047:Ovalbumin