Transfection of ram spermatozoa with pEGFP carrying human lysozyme gene


1 Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran-Iran

2 Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran-Iran

3 Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran-Iran.

4 Graduated From the Faculty of Veterinary Medicine, University of Tehran, Tehran-Iran


BACKGROUND: As we all know, sperm has the capacity to take up foreign DNA, therefore, sperm mediated gen transfer can be an inexpensive and simple method in animal transgenesis in various species. However, there is not sufficient evidence of DNA uptake by ovine spermatozoa. Objectives: The purpose of the present study was to examine the uptake of human lysozyme gene contained plasmid (pEGFP-IRES-hLys) by ovin spermatozoa. Methods: In the first experiment, semen was prepared from three ram (each ram two times) by electrical method. After removal of seminal plasma, 1x106 spermatozoa were incubated with rhodamin-labled pEGFP-IRES-hLys in TCM199 for 15, 30, 60 and 120 minutes and then observed for motility, uptake percent and uptake intensity by florescent microscopy. Also after 60 minutes incubation sperms were treated by DNaseI to assay adsorption or uptake of pEGFP-IRES-hLys. In the second experiment, washed and unwashed sperms were incubated with rhodamin-labled pEGFP-IRES-hLys in TCM199 for 30 and 60 minutes to evaluate the effect of presence seminal plasma on sperm uptake and motility. Results: The findings showed that increasing incubation time increased number/percentage of spermatozoa carrying exogenous DNA and its intensity. But this different was significant only up to 30 minutes. We found that 60.16% of the cells were bound to DNA after 120 minutes incubation. Incubation with exogenous DNA induced a slightly decrease in sperm total and progressive motility. But no post acrosom uptaked sperm was motile. After treatment with DNaseI, strong florescent emission from post acrosom indicated absorption of pEGFP-IRES-hLys by spermatozoa. Presence of seminal plasma induced a slightly decrease in percent of DNA absorbed spermatozoa and absorption intensity, but did not inhibit completely. ConclusionS: Ram spermatozoa showed a high capacity to bind DNA quickly and reach a maximum after 30 min. However, no sperm with real uptake (post acrosomal) was motile. Incubation with lower DNA concentration and/or shorter time may be helpful.