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

Abstract

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 1 h: 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 20 min and heated for 2 min 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 < 0.05) by 29.9% in heat shock group compared to the control. Adding DMSO improved (P < 0.05) the total and progressive motilities by 8.2% and 19.8%, respectively in heat shock group compared to the heat shock group without DMSO. The results documented the ability of ovine spermatozoa to uptake the exogenous vector. Also, sperm incubation with 3% DMSO is the best method to introduce the exogenous vector into spermatozoa without notable adverse effects on sperm motilities.

A review of transgenic animal techniques and their applications

“…Transgenic mice, rabbits, pigs, sheep, cows, chickens, and fish have been created by incubating sperm cells with foreign DNA and fertilizing them in vitro or in vivo [ 9 , 10 ]. Furthermore, this operation does not necessitate any special equipment or skills, and it may be carried out in the field.…”

“…Furthermore, this operation does not necessitate any special equipment or skills, and it may be carried out in the field. Another fascinating feature of using sperm as DNA vectors is the concept of mass transgenesis [ 9 , 10 ]. In subsequent studies, the successful introduction of the exogenous GH expression vector into the sperm head allowed for the production of GH-transgenic sheep characterized by a high growth rate in order to reduce the meat shortage in Egypt [ 10 ].…”

“…Fresh semen is taken from donor animals and cleaned several times before being centrifuged to remove seminal plasma. Animal artificial insemination, incubation of sperm cell suspensions with foreign plasmid DNA (about 1 h at 18), and dilution in suitable extender dimethyl sulfoxide (DMSO) and Triton X-100, a mild polar detergent, were used to improve DNA uptake in sperm [ 9 , 10 ]. The sperm membrane was destabilized as a result, allowing foreign DNA full access to the sperm.…”