PMID- 21775957
OWN - NLM
STAT- MEDLINE
DCOM- 20120418
LR  - 20231213
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 53
DP  - 2011 Jul 8
TI  - Optimized protocol for efficient transfection of dendritic cells without cell 
      maturation.
PG  - e2766
LID - 2766 [pii]
LID - 10.3791/2766 [doi]
AB  - Dendritic cells (DCs) can be considered sentinels of the immune system which play 
      a critical role in its initiation and response to infection. Detection of 
      pathogenic antigen by naive DCs is through pattern recognition receptors (PRRs) 
      which are able to recognize specific conserved structures referred to as 
      pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs 
      triggers an intracellular signaling cascade resulting in their activation and 
      transformation to mature DCs. This process is typically characterized by 
      production of type 1 interferon along with other proinflammatory cytokines, 
      upregulation of cell surface markers such as MHCII and CD86 and migration of the 
      mature DC to draining lymph nodes, where interaction with T cells initiates the 
      adaptive immune response. Thus, DCs link the innate and adaptive immune systems. 
      The ability to dissect the molecular networks underlying DC response to various 
      pathogens is crucial to a better understanding of the regulation of these 
      signaling pathways and their induced genes. It should also help facilitate the 
      development of DC-based vaccines against infectious diseases and tumors. However, 
      this line of research has been severely impeded by the difficulty of transfecting 
      primary DCs. Virus transduction methods, such as the lentiviral system, are 
      typically used, but carry many limitations such as complexity and bio-hazardous 
      risk (with the associated costs). Additionally, the delivery of viral gene 
      products increases the immunogenicity of those transduced DCs. Electroporation 
      has been used with mixed results, but we are the first to report the use of a 
      high-throughput transfection protocol and conclusively demonstrate its utility. 
      In this report we summarize an optimized commercial protocol for high-throughput 
      transfection of human primary DCs, with limited cell toxicity and an absence of 
      DC maturation. Transfection efficiency (of GFP plasmid) and cell viability were 
      more than 50% and 70% respectively. FACS analysis established the absence of 
      increase in expression of the maturation markers CD86 and MHCII in transfected 
      cells, while qRT-PCR demonstrated no upregulation of IFNbeta. Using this 
      electroporation protocol, we provide evidence for successful transfection of DCs 
      with siRNA and effective knock down of targeted gene RIG-I, a key viral 
      recognition receptor, at both the mRNA and protein levels.
FAU - Bowles, Robert
AU  - Bowles R
AD  - Center for Translational Systems Biology and Department of Neurology, Mount Sinai 
      School of Medicine, USA.
FAU - Patil, Sonali
AU  - Patil S
FAU - Pincas, Hanna
AU  - Pincas H
FAU - Sealfon, Stuart C
AU  - Sealfon SC
LA  - eng
GR  - HHSN2662000500021C/PHS HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Video-Audio Media
DEP - 20110708
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 0 (RNA, Small Interfering)
RN  - 0 (Receptors, Immunologic)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - 77238-31-4 (Interferon-beta)
RN  - EC 3.6.1.- (RIGI protein, human)
RN  - EC 3.6.4.13 (DEAD Box Protein 58)
RN  - EC 3.6.4.13 (DEAD-box RNA Helicases)
SB  - IM
MH  - Cell Separation/methods
MH  - DEAD Box Protein 58
MH  - DEAD-box RNA Helicases/genetics
MH  - Dendritic Cells/immunology/*physiology
MH  - Flow Cytometry/methods
MH  - Gene Knockdown Techniques/*methods
MH  - Green Fluorescent Proteins/biosynthesis/genetics
MH  - Humans
MH  - Interferon-beta/biosynthesis/genetics
MH  - Plasmids/genetics
MH  - RNA, Small Interfering/administration & dosage/genetics
MH  - Receptors, Immunologic
MH  - Transfection/*methods
PMC - PMC3196177
EDAT- 2011/07/22 06:00
MHDA- 2012/04/19 06:00
PMCR- 2013/07/08
CRDT- 2011/07/22 06:00
PHST- 2011/07/22 06:00 [entrez]
PHST- 2011/07/22 06:00 [pubmed]
PHST- 2012/04/19 06:00 [medline]
PHST- 2013/07/08 00:00 [pmc-release]
AID - 2766 [pii]
AID - 10.3791/2766 [doi]
PST - epublish
SO  - J Vis Exp. 2011 Jul 8;(53):e2766. doi: 10.3791/2766.