Exogenous DNA length and quantity affect the transfection rate, but not sperm viability during Sperm-Mediated Gene Transfer

Autor(es)

Feitosa, Weber Beringui ; Ilazzotto, Marcella Pecora ; Mendes, Camilla Mota ; da Rocha, Andre Monteiro ; Avanzo, Jose Luis ; Martins, Elizabeth Angelica Leme ; Visintin, Jose Antonio ; Assumpcao, Mayra Elena Ortiz D'Avila

• Tipo de documento: Artigo Científico
• Fonte: GENE REPORTS; v. 29, p. 7-pg., 2022-09-11.

Resumo

Spermatozoa have a spontaneous ability to take up exogenous DNA in a process regulated by specific mecha-nisms. This ability has been used to carry exogenous DNA into oocytes during fertilization to produce transgenic animals; a process called sperm-mediated gene transfer (SMGT). However, it is still an inefficient method and little is known about the effect of exogenous DNA once associated with spermatozoa, on sperm characteristics. Therefore, the objective of the present work was to evaluate the effects of exogenous DNA length and its amount on DNA uptake by bovine spermatozoa as well as spermatozoa viability. For that, spermatozoa (5 x 106 cells/ mL) were incubated for 1 h at 38.5 degrees C with different exogenous DNA lengths (2.2, 5.5, or 8.5 kb) at different concentrations (number of molecules or ng). The association of exogenous DNA with spermatozoa was quantified by PCR real-time and the spermatozoa viability was evaluated by flow cytometry. Here, we show that no matter the amount of exogenous DNA used, larger sequences are less efficiently (p < 0.05) associated with bovine spermatozoa. Besides that, the length and amount of exogenous DNA do not compromise sperm viability. Taken together, the results support that the length of exogenous DNA is more important than the amount used to in-fluence its association with sperm cells. Thus, the size and quantity of exogenous DNA can be optimized to in-crease SMGT protocols, without altering the sperm viability. (AU)

• Processo FAPESP: 03/10234-7 - Espermatozoide bovino como vetor de transgene: efeito da estrutura e do tamanho do dna exogeno na internalizacao no nucleo do espermatozoide.
• Beneficiário: Weber Beringui Feitosa
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• Processo FAPESP: 03/07456-8 - Estudo da integracao do dna exogeno em espermatozoides de bovinos.
• Beneficiário: Mayra Elena Ortiz D' Avila Assumpcao
• Modalidade de apoio: Auxílio à Pesquisa - Regular