Sperm-mediated gene transfer: effect on bovine in vitro embryo production.
The technique of sperm-mediated gene transfer (SMGT) can be used to delivery exogenous DNA into the oocyte. However, it has low repeatability and produces inconsistent results. In order to optimize this technique, it is necessary to study the mechanism by which DNA enters the sperm cell and integrates in the sperm genome. Furthermore, studies must focus in the maintenance of sperm cell viability and function. The aim of this study was to evaluate different SMGT protocols of sperm electroporation or capacitation (CaI) aiming to maintain sperm viability in the production of bovine embryos in vitro. Frozen-thawed semen was divided in two experimental groups (electroporation or CaI) and one control group (non-treated cells). For the electroporation method, five different voltages (100, 500, 750, 1000 or 1500 V) with 25 μF capacitance were used. For CaI treatment, combinations of two CaI concentrations (250 nM or 500 nM), two incubation periods of sperm cells with CaI (1 or 5 min) and two incubation periods that mimicked time of sperm cell interaction with exogenous DNA molecules (1 or 2 h) were evaluated. According to our data, electroporation and CaI treatments do not prevent sperm penetration and oocyte fertilization and can be an alternative method to achieve satisfactory DNA delivery in SMGT protocols.
Centro de Ciências Naturais e Humanas, Federal University of ABC (UFABC); Santo André-SP; Brazil.
Renata
R
Simões
Alessandra Corallo
AC
Nicacio
Mario
M
Binelli
Fabiola Freitas
FF
de Paula-Lopes
Marcella Pecora
MP
Milazzotto
José Antonio
JA
Visintin
Mayra Elena Ortiz
ME
D'Ávila Assumpção
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D061207:Calcium Ionophores / Q000494:pharmacology; D002417:Cattle; D002470:Cell Survival; D004247:DNA / Q000008:administration & dosage*; D018274:Electroporation; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D047108:Embryonic Development; D005307:Fertilization in Vitro; D018014:Gene Transfer Techniques; D066298:In Vitro Techniques; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013081:Sperm Motility; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*
D061207:Calcium Ionophores; D000001:Calcimycin; D004247:DNA