Construction of ovine GH-pmKate2N expression vector and its uptake by ovine spermatozoa using different methods

• Published in: Journal of Genetic Engineering and Biotechnology 2017-06, Vol.15 (1), p.13-21
• Main Authors: Shakweer, W.M.E., Hafez, Y.M., El-Sayed, A.A., Awadalla, I.M., Mohamed, M.I.
• Format: Article
• Language: English
• Subjects: I : Animal Biotechnology; Ovine growth hormone; pmKate2-N expression vector; Sperm uptake
• ISSN: 1687-157X; 2090-5920
• DOI: 10.1016/j.jgeb.2017.04.001

This study aims to produce transgenic ovine spermatozoa bearing Ossimi sheep growth hormone (Os_GH) cDNA using different methods. The complete coding sequence of Os_GH has been registered in GenBank accession no. KP221575. The sequence of Os_GH cDNA has been subcloned into pmkate2-N expression vectors to construct Os_GH-pmKate2-N vector. Five groups of sperm uptake were submitted. All groups were incubated at 37°C for 1h: Control (sperm cells were incubated without vector), Traditional incubation (sperm cells were incubated with vector), Heat shock (sperm cells were incubated with vector at 4°C for 20min and heated for 2min at 42°C), Heat shock+Dimethyl sulfoxide (DMSO) (sperm cells were incubated with vector and supplemented with 3% of DMSO and then submitted to heat shock regime) and DMSO (sperm cells were incubated with vector and supplemented with 3% DMSO). The sperm genomic DNA in groups was extracted. The Os_GH-pmKate2-N vector was introduced efficiently into the head of sperm cells in all treated groups. Adding DMSO either with or without heat shock increased the sperm uptake. The progressive motility was reduced (P