1. Efficiency and cell viability implications using tip type electroporation in zebrafish sperm cells
- Author
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Leandro S. Nunes, Antonio Sergio Varela, Izani Bonel Acosta, Gabriela T. Rassier, Charles Nunes Froes, William Borges Domingues, Eduardo B. Blödorn, Eliza Rossi Komninou, Eduardo N. Dellagostin, Carine Dahl Corcini, Vinicius Farias Campos, Larissa O. Daneluz, and Amanda Weege Da Silveira Martins
- Subjects
Male ,0301 basic medicine ,animal structures ,Cell Survival ,Fertilization in Vitro ,Semen analysis ,Transfection ,Exogenous DNA ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,medicine ,Animals ,Viability assay ,Molecular Biology ,Zebrafish ,Danio rerio ,medicine.diagnostic_test ,biology ,urogenital system ,Chemistry ,Electroporation ,fungi ,Gene Transfer Techniques ,SMGT ,DNA ,General Medicine ,biology.organism_classification ,Spermatozoa ,Sperm ,Mitochondria ,Cell biology ,Transgenesis ,030104 developmental biology ,030220 oncology & carcinogenesis ,embryonic structures ,Sperm Motility ,Original Article ,Reactive Oxygen Species - Abstract
Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, transfection into fish sperm cells still needs to be improved. The objective was to demonstrate the feasibility of tip type electroporation in zebrafish sperm, showing a protocol that provide high transfection efficiency, with minimal side-effects. Sperm was transfected with a Cy3-labelled DNA using tip type electroporation with voltages ranging from 500 to 1500 V. Sperm kinetics parameters were assessed using Computer Assisted Semen Analysis (CASA) and cell integrity, reactive oxygen species (ROS), mitochondrial functionality and transfection rate were evaluated by flow cytometry. The transfection rates were positively affected by tip type electroporation, reaching 64.9% ± 3.6 in the lowest voltage used (500 V) and 86.6% ± 1.9 in the highest (1500 V). The percentage of overall motile sperm in the electrotransfected samples was found to decrease with increasing field strength (P 0.05). Overall results indicate that tip type electroporation enhances the internalization of exogenous DNA into zebrafish sperm cells with minimal harmful effects to sperm cells. Electronic supplementary material The online version of this article (10.1007/s11033-020-05658-2) contains supplementary material, which is available to authorized users.
- Published
- 2020