Uptake of exogenous bovine GH–pmKate2–N expression vector by rams spermatozoa

Abstract

Background: Sperm-mediated gene transfer (SMGT) is a technique that utilizes the ability of the spermatozoa to take up exogenous DNA. Growth hormone is anabolic hormone that plays an important role in muscle-building process and milk production in all animals. High blood concentration of growth hormones (GH) was observed for animals that were genetically selected for high milk production or for low carcass fatness levels. The present study aimed to investigate and enhance the capacity of ovine spermatozoa to uptake exogenous growth hormone cDNA and its impact on sperm motility. The current study is an introduction for further future studies to produce transgenic Egyptian sheep characterized with high productive performance. Methods: The growth hormone cDNA sequence was extracted from pituitary gland of Egyptian × Holstein (EH_ GH) cattle and subcloned into the pmKate2-N vector to construct the EH_GH-pmKate2-N expression vector. The complete sequence of EH_GH mRNA was registered in GenBank (AC: KP221576). A total of three groups were assessed for the sperm uptake experiment, namely, negative control, positive control, and dimethyl sulfoxide (DMSO) groups; all treated groups were incubated with the EH_GH-pmKate2-N vector. The expression of EH_GH protein was detected in DH10B cells using a fluorescence microscope and the SDS polyacrylamide gel electrophoresis. Results: The EH_GH-pmKate2-N vector was expressed in cultured Escherichia coli cells, and the molecular weight of EH_GH protein was 24,558 Da. The EH_GH-pmKate2-N vector was introduced efficiently into the heads of the spermatozoa in the DMSO and positive control groups. Incubation of the spermatozoa with the vector caused a significant reduction in progressive motility compared to the negative control. Conclusion: The present results demonstrated the ability of ovine spermatozoa to take up the exogenous vector without notable deleterious effects on sperm motility. In subsequent studies, the successful introduction of the exogenous GH expression vector into the sperm head allows for the production of GH-transgenic sheep characterized by a high growth rate in order to reduce the meat shortage in Egypt.

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 ].…”

“…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 ]. The main binding site of foreign DNA in mouse sperm is mediated by a complex structure of molecules from the class 2 major histocompatibility complex, which is found in the posterior area of the sperm head, according to Wu et al [ 34 ].…”

“…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.…”