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 introduc e new genes, but in vivo neuronal transfection has posed difficulties in th e mature intact nervous system of vertebrates, including the lamprey, In th is paper we report successful transfection of neurons in the brain of livin g lampreys by means of a hand-held Helios Gene Gun. Particle-mediated ("gen e gun") gene transfer has been applied to a variety of cell and tissue type s but although it has been used in brain slices and dissociated cultured ne urons, to our knowledge it has not been reported as a method for transfecti on of brain cells in a living animal. Gold particles coated with plasmids c ontaining the gene for the reporter P-galactosidase were propelled by heliu m at 150-200 psi toward the exposed floor of the 4th ventricle, Transfected animals were examined by X-gal histochemistry at various recovery times. b eta -glactosidase activity was detected as early as 2 days after gene trans fer and lasted for at least 6 weeks, the longest time studied. Transgene ex pression 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 th e possibility of identifying the growth cones of developing or regenerating axons belonging to transfected neurons in functional studies of manipulate d genes. (C) 2001 Academic Press.