Factors affecting porcine sperm mediated gene transfer.
"Sperm mediated gene transfer" (SMGT) is based on the ability of sperm cells to bind exogenous DNA. The main objective of this study was to improve the production of transgenic pigs by SMGT. Taking into account that there is a lack of repeatability in studies of SMGT and that the mechanism of binding and internalization of exogenous DNA is a question that has not been solved, different factors involved in the production of transgenic animals by SMGT method were evaluated. Here we set out to: (1) evaluate the sperm capacity to bind exogenous DNA after DMSO treatment; (2) determine the location of the transgene-spermatozoa interaction; and (3) evaluate the efficiency of production of transgenic piglets by deep intrauterine artificial insemination (AI) with sperm incubated with DNA. The percentage of DNA binding was higher than 30% after 2h of co-culture, but it was not affected by sperm treatment with DMSO (0.3% or 3%). The integrity of the sperm plasma membrane plays a critical role in DNA interaction, and altered plasma membranes facilitate interactions with exogenous DNA. DNA bound mainly to spermatozoa with reduced viability. DNA molecules were found to be mainly associated to the post-acrosomal region (61.9%). After deep intrauterine AI a total of 29 piglets were obtained, but none of them integrated the transgene. In conclusion, although it has been confirmed that DNA can associate with boar spermatozoa, the efficiency of producing transgenic pigs by AI was not confirmed by the present experiments, mainly due to a reduced DNA binding to functional spermatozoa.
García-Vázquez
Francisco Alberto
FA
Departamento de Fisiología, Facultad de Veterinaria, Universidad de Murcia, 30100 Murcia, Spain. fagarcia@um.es
Ruiz
Salvador
S
Grullón
Luis Alberto
LA
de Ondiz
Aitor
A
Gutiérrez-Adán
Alfonso
A
Gadea
Joaquín
J
D000818:Animals; D030801:Animals, Genetically Modified; D004247:DNA / Q000235:genetics; D005260:Female; D018014:Gene Transfer Techniques / Q000662:veterinary*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007315:Insemination, Artificial / Q000662:veterinary; D008297:Male; D011247:Pregnancy; D013094:Spermatozoa / Q000502:physiology*; D013552:Swine / Q000235:genetics* / Q000502:physiology
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA