Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA.
Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratically thereafter for up to 2 months. Phenotypic analysis showed that EGFP+ ALDC expressed MHC class II, WC6, CD1b, and SIRPalpha markers. Plasmid, detected by PCR, was found in lymph cells and cell-free plasma on a daily basis, and was present variably for up to 2 months. Plasmid was also detected in purified CD1b+ ALDC, but the presence of plasmid did not correlate with EGFP expression by ALDC. Free EGFP in afferent lymph plasma was detectable by luminometry only after three administrations of the plasmid. The results show that gene gun administered pEGFP persisted for extended periods after a single administration, leeching out of skin on a daily basis. The plasmid was associated with both the cellular and fluid components of afferent lymph. EGFP protein appeared in afferent lymph in a pulsatile manner, but associated only with ALDC.
Watkins
Craig
C
Division of Veterinary Biomedical Sciences, University of Edinburgh, Easter Bush Veterinary Centre, Easter Bush, Midlothian EH25 9RG, UK.
Hopkins
John
J
Harkiss
Gordon
G
D000818:Animals; D004247:DNA / Q000737:chemistry; D003713:Dendritic Cells / Q000276:immunology*; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D007202:Indicators and Reagents / Q000737:chemistry; D007271:Injections, Intradermal / Q000662:veterinary; D008196:Lymph / Q000166:cytology; D010957:Plasmids / Q000235:genetics*; D012756:Sheep; D012867:Skin / Q000276:immunology*; D014611:Vaccination / Q000662:veterinary*; D019444:Vaccines, DNA / Q000276:immunology
D007202:Indicators and Reagents; D019444:Vaccines, DNA; D049452:Green Fluorescent Proteins; D004247:DNA