First study of sperm mediated gene transfer in Egyptian river buffalo

Mohamed S Hassanane; Aida I El Makawy; Sahar M Helalia; Ahmed S Abdoon; Kamal M A Khalil; Tharwat A Ghanem; Amany M Tohamy; Xiao-Feng Sun; Wei Shen

doi:10.1016/j.jgeb.2017.06.003

First study of sperm mediated gene transfer in Egyptian river buffalo

J Genet Eng Biotechnol. 2017 Dec;15(2):475-482. doi: 10.1016/j.jgeb.2017.06.003. Epub 2017 Jun 22.

Authors

Mohamed S Hassanane¹, Aida I El Makawy¹, Sahar M Helalia¹, Ahmed S Abdoon², Kamal M A Khalil³, Tharwat A Ghanem⁴, Amany M Tohamy⁴, Xiao-Feng Sun⁵, Wei Shen⁵

Affiliations

¹ Cell Biology Department, Genetic Engineering and Biotechnology Division, National Research Centre, Egypt.
² Animal Reproduction and Artificial Insemination Department, Veterinary Research Division, National Research Centre, Egypt.
³ Genetics and Cytology Dept., Genetic Engineering and Biotechnology Division, National Research Centre, Egypt.
⁴ Zoology Department, Faculty of Science, Helwan University, Egypt.
⁵ Institute of Reproductive Sciences, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China.

Abstract

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 20 µg/ml from the linarized DNA, for 15 min at 4 °C are the best conditions to produce transgenic buffalo embryo using sperm mediated gene transfer.

Keywords: ANOVA, analysis of variance; Buffalo embryos; CCC, covalently closed circular; COCs, Cumulus oocyte complexes; DMSO; DMSO, Dimethyle sulphoxide; EGFP; EGFP, enhanced green fluorescent protein; IVF, in vitro fertilization; MII, second meiotic division; OD, optical density; PBS, Phosphate buffer saline; SMGT; SMGT, sperm mediated gene transfer; TCM199, tissue culture medium; Transgenic.