Effects of electropermeabilization on the association of foreign DNA with pig sperm.
This study was conducted to evaluate the effects of an electric pulse (electroporation/electropermeabilization) on the binding of foreign DNA molecules to porcine spermatozoa. We previously examined various parameters involved in the association of foreign DNA with sperm after a simple incubation procedure and now report the effects of electroporation on this association. Using end-labeled and random primer labeled lambda HindIII DNA fragments (23-0.125 kb), it was demonstrated that the DNA fragments interacted with the sperm after electroporation. These samples were then centrifuged and washed extensively to establish if any of the labeled DNA was associated with the spermatozoa. It was determined that approximately 10(8) molecules of DNA were associated with 1.5 x 10(7) mL-1 motile spermatozoa after five medium washes. After each wash, samples were withdrawn for gel analysis and scintillation counting. Gel analysis followed by autoradiography revealed the distinctive band pattern of lambda HindIII DNA associating with sperm. In situ visualization studies with biotin-labeled DNA revealed that approximately 75% of motile sperm carried DNA bound to the post-acrosomal region. However, the intensity of the binding varied, with some sperm being more strongly stained than others. Using [3H]dCTP-labeled DNA followed by light microscope autoradiography, approximately 70% of the sperm were strongly stained in the post-acrosomal region. There was a 5-10% increase in the amount of DNA bound by sperm when the samples were electroporated.
Horan
R
R
Department of Microbiology, University College, Galway, Ireland.
Powell
R
R
Bird
J M
JM
Gannon
F
F
Houghton
J A
JA
D000818:Animals; D030801:Animals, Genetically Modified; D001345:Autoradiography; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004560:Electricity; D008297:Male; D009693:Nucleic Acid Hybridization; D013094:Spermatozoa / Q000648:ultrastructure*; D013552:Swine; D014162:Transfection
D004247:DNA