First study of sperm mediated gene transfer in Egyptian river buffalo

• Published in: Journal of Genetic Engineering and Biotechnology Vol. 15; no. 2; pp. 475 - 482
• Main Authors: Hassanane, Mohamed S., El Makawy, Aida I., Helalia, Sahar M., Abdoon, Ahmed S., Khalil, Kamal M.A., Ghanem, Tharwat A., Tohamy, Amany M., Sun, Xiao-Feng, Shen, Wei
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2017; Academy of Scientific Research and Technology, Egypt; Elsevier
• Subjects: Animal Biotechnology; Buffalo embryos; DMSO; EGFP; SMGT; Transgenic; PBS; SMGT; CCC; COCs; DMSO; Buffalo embryos; Transgenic; EGFP; MII; OD; ANOVA; IVF; TCM199; EGFP, enhanced green fluorescent protein; TCM199, tissue culture medium; OD, optical density; COCs, Cumulus oocyte complexes; DMSO, Dimethyle sulphoxide; PBS, Phosphate buffer saline; CCC, covalently closed circular; SMGT, sperm mediated gene transfer; IVF, in vitro fertilization; ANOVA, analysis of variance; MII, second meiotic division
• ISSN: 1687-157X; 2090-5920
• DOI: 10.1016/j.jgeb.2017.06.003

The present study was carried out to find the best treatments for enhancing the ration of insertion of a desired gene construct (pEGFP-N1) onto the sperm of buffalo as the first step for the production of transgenic buffalo using sperm mediated gene transfer (SMGT). The tested conditions were plasmid DNA concentration, sperm concentration, transfecting agent concentration: Dimethyle sulphoxide (DMSO) and time of transfection. The study proved that the best conditions for producing transgenic embryos were incubation sperm solution its concentration is 107/ml sperm with 3% DMSO: with 20µg/ml from the linarized DNA, for 15min at 4°C are the best conditions to produce transgenic buffalo embryo using sperm mediated gene transfer.