In vivo transfection of lamprey brain neurons by gene gun delivery of DNA

The lamprey has been used extensively in studies of CNS axon regeneration. Progress in determining molecular mechanisms involved in regeneration will require the ability to manipulate expression of target genes or to introduce new genes, but in vivo neuronal transfection has posed difficulties in the mature intact nervous system of vertebrates, including the lamprey, In this paper we report successful transfection of neurons in the brain of living lampreys by means of a hand-held Helios Gene Gun. Particle-mediated ("gene gun") gene transfer has been applied to a variety of cell and tissue types but although it has been used in brain slices and dissociated cultured neurons, to our knowledge it has not been reported as a method for transfection of brain cells in a living animal. Gold particles coated with plasmids containing the gene for the reporter P-galactosidase were propelled by helium at 150-200 psi toward the exposed floor of the 4th ventricle, Transfected animals were examined by X-gal histochemistry at various recovery times. beta -glactosidase activity was detected as early as 2 days after gene transfer and lasted for at least 6 weeks, the longest time studied. Transgene expression lasted longer in neurons than in glia, The expression product was transported anterogradely into reticulospinal axone and by 6 weeks could be traced into the spinal cord for 8-10 mm caudal to the obex, This raises the possibility of identifying the growth cones of developing or regenerating axons belonging to transfected neurons in functional studies of manipulated genes. (C) 2001 Academic Press.