Spontaneous uptake of exogenous DNA by goat spermatozoa and selection of donor bucks for sperm-mediated gene transfer
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Sperm-mediated gene transfer (SMGT) has been long heralded as a faster and cheaper alternative to more commonly used methods of producing transgenic animals. In this study, the capra semen ejaculates were pooled together and then incubated in vitro with DIG-labeled DNA. The binding and internalizing rates were observed by the in situ hybridization methods. We also compared the standard sperm parameters and the efficiencies of interaction with exogenous DNA of 60 individuals to select donor bucks for SMGT. It was showed that labeled exogenous DNA was detected in different localizations in spermatozoa but genuine DNA uptake, in contrast to mere binding, seems to be limited to the postacrosomal region. The removal of seminal plasma increased significantly (P < 0.01) the capability in picking up exogenous DNA. Use of frozen-thawed semen (without cryoprotectant agents) and Triton X-100 treatment also increased significantly (P < 0.01) the DNA-binding capacity, but reduced the sperm viability. The binding rates (the proportion of labeled-DNA positive spermatozoa to all the spermatozoa counted) of 60 buck individuals were in the range of 3.08–73.39%, and the internalizing rates (the proportion of DNaseI-treated labeled-DNA positive spermatozoa to all the spermatozoa counted) were 4.83–70.00%. About 8.34% (5/60) bucks showed high binding, but low internalizing ability. Chi-square test showed that there was significant difference among the breeds (x 2 = 26.515, P < 0.01). Eight individual bucks that demonstrated high DNA uptake were selected for SMGT. It was demonstrated that the goat spermatozoa was capable of spontaneous uptake of exogenous DNA. Seminal fluid inhibits DNA uptake and that membrane disruption increases DNA binding but greatly diminishes uptake.
KeywordsSperm-mediated gene transfer Sperm Exogenous DNA Seminal plasma Goat
The work was supported by: national science and technology specific project, grant number: 2008ZX08008-004, the Fundamental Research Funds for the Central Universities, grant number: XDJK2010C092 and the national natural science foundation of China, grant number: 30600430. We thank Dr. S. G. Revell (Genus Breeding Ltd. Ruthin, Denbighshire LL15 2UP, UK) and Mrs. Aparna Udupi (Department of Genetics and Biotechnology, Research Centre Foulum, Aarhus University, DK-8830 Tjele, DK) for critical reading the manuscript. We also thank seven anonymous reviewers and editors for constructive and professional comments.
- 14.Zhao Y et al (2010) Production of transgenic goats by sperm-mediated exogenous DNA transfer method. Asian-Aust J Anim Sci 23(1):33–40Google Scholar
- 16.Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 271–275Google Scholar
- 17.Zhao Y-J, Li F-B, Sun X-M, Zhou J-H, Wei H (2005) Age- and- breed-dependence of exogenous DNA binding to spermatozoa in goats. Acta Zoologica Sinica 51(6):1058–1066Google Scholar
- 31.Zhao Y, Wang Y, Wang J, Li F, Wei H (2008) Effect of cryopreservation on the efficiencies of goat sperm in picking up exogenous DNA and resulted transgenic embryos. Acta Zoologica Sinica 54(6):1089–1097Google Scholar
- 32.Shim SW, Kim YH, Lee HT, Shim H (2008) Effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and transgenesis of porcine embryos produced by intracytoplasmic sperm injection. Asian-Aust J Anim Sci 21(3):358–363Google Scholar