Authors : Shun Zhang, Jin-Yue Shi, Xiao-Mei Guan, Jin-Ji Yang, Ying-Ming Wei, Qing-You Liu, De-Shun Shi and Feng-Hua Lu
Abstract: Cytoplasmic injection of exogenous DNA was a high potential methodology that would tremendously increase the efficiency of transgene. In the present study, researchers take a new look at the ancient method and investigate the influencing factors on embryonic development and transgenic efficiency following cytoplasmic injection using pEGFP-N1 plasmid. In the first experiment, researchers evaluated embryonic development and EGFP expression in embryos produced by cytoplasmic injection with different concentrations of pEGFP-N1 plasmid (30, 45, 60 and 75 ng μL-1) and found that the concentration of 60 ng μL-1 was optimal for increasing in both blastocysts rate and EGFP-positive blastocysts rate. In the second experiment, the embryonic development and EGFP expression after cytoplasmic injection with different volumes (40, 50 and 60 pL) of exogenous DNA was investigated, the result showed that the optimal volume of injected was 40 pL with which approximately 54.5% of the blastocysts rate and 59.3% of the blastocysts expression EGFP were obtained. Finally, in order to find out the most appropriate timing for cytoplasmic injection, three time periods (16, 18 and 20 h) were assayed to harvest the zygotes. After being injected and cultured, the result confirmed that 16 h group (68.6%) had a significant differences compare to 18 h group (44.9%) and 20 h group (9.7%) at EGFP-positive blastocyst rate but there was no significant differences as to blastocyst rate among these three groups. Two pups were given birth after embryo transplantation. The integration of the foreign DNA was confirmed by PCR and an expected 720 bp products showed that both of the two pup’s tissues including kidneys and livers carry EGFP gene. The results of frozen tissue sections analyzed by confocal laser scanning microscope and western blot also were confirmed that EGFP expression in kidney and liver. In conclusion, the results of this study imply that the method of cytoplasmic injection at one-cell stage is feasible for producing transgenic animals which can significantly improve the transgenic efficiency.
Shun Zhang, Jin-Yue Shi, Xiao-Mei Guan, Jin-Ji Yang, Ying-Ming Wei, Qing-You Liu, De-Shun Shi and Feng-Hua Lu, 2013. Factors Affecting the Efficiency of Introducing Foreign DNA into Rabbit Zygotes by Cytoplasmic Injection. Journal of Animal and Veterinary Advances, 12: 897-903.