DNA transfection of mononuclear cells in muscle tissue.
BACKGROUND
Genes encoding non-self proteins may be injected into skeletal muscles in vivo to obtain induction of cellular and humoral immune responses against the encoded antigens (DNA vaccination). Bone marrow derived professional antigen-presenting cells (APCs) play a key role in the induction of immunity by DNA vaccination. In the present work we have investigated whether the APCs are transfected by DNA injection into muscle.


METHODS
DNA encoding green fluorescent protein (GFP) was injected into rat and mouse limb muscle and followed by electroporation. Whole mount muscle tissue with GFP-positive mononuclear cells (MNCs) were treated with immunocytochemical markers specific for leukocytes, and studied with fluorescent microscopy. To detect transfected cells migrating to peripheral lymphoid tissue RT-PCR was applied on RNA isolated from the draining popliteal lymph node and spleen. Lymphoid tissue was also analyzed with real-time PCR for distribution of the injected plasmid.


RESULTS
MNCs were transfected after intramuscular DNA injection, and, following DNA injection with electroporation, the number of GFP-positive MNCs increased 6-fold in rats and 14-fold in mice. None of the GFP-positive MNCs were stained with leukocyte-specific antibodies. Even though GFP encoding DNA was detected in the popliteal lymph node, no RNA encoding GFP was found in the lymph node or spleen. However, MHC II-positive cells in the muscle tissue appeared preferentially around the transfected MNCs.


CONCLUSIONS
Many MNCs in the muscle are transfected after intramuscular DNA injection. Electroporation significantly increases the number of transfected MNCs. None of the observed transfected MNCs however were leukocytes. MHC II-positive cells accumulated around transfected MNCs; this suggests that transfer of antigen from transfected MNCs to APCs may contribute to the immune response.
Grønevik
Eirik
E
Department of Physiology, University of Oslo, Box 1103 Blindern, 0316 Oslo, Norway.
Tollefsen
Stig
S
Immunological institute, Rikshospitalet, 0027 Oslo, Norway.
Sikkeland
Liv Ingunn Bjoner
LIB
Centre for Occupational and Environmental Medicine, Rikshospitalet, 0027 Oslo, Norway.
Haug
Terje
T
Centre for Occupational and Environmental Medicine, Rikshospitalet, 0027 Oslo, Norway.
Tjelle
Torunn Elisabeth
TE
Inovio AS, Oslo Research Park, Gaustadalleen 21, 0349 Oslo, Norway.
Mathiesen
Iacob
I
Inovio AS, Oslo Research Park, Gaustadalleen 21, 0349 Oslo, Norway.
D000818:Animals; D004247:DNA / Q000235:genetics*; D018274:Electroporation; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins; D007273:Injections, Intramuscular; D008164:Luminescent Proteins; D051379:Mice; D009132:Muscles / Q000166:cytology*; D051381:Rats; D014162:Transfection / Q000379:methods*
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA