@article{MAKHILLJAVA20131284114,
    title = {Factors Affecting the Efficiency of Introducing Foreign DNA into Rabbit Zygotes by Cytoplasmic Injection},
    journal = {Journal of Animal and Veterinary Advances},
    volume = {12},
    number = {8},
    pages = {897-903},
    year = {2013},
    issn = {1680-5593},
    doi = {javaa.2013.897.903},
    url = {https://makhillpublications.co/view-article.php?issn=1680-5593&doi=javaa.2013.897.903},
    author = {De-Shun,Shun,Jin-Yue,Xiao-Mei,Jin-Ji,Ying-Ming,Qing-You and},
    keywords = {Rabbit zygote,cytoplasmic injection,transgenic efficiency,pEGFP-N1,animals},
    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 &#956;L<SUP>-1</SUP>) and found that the concentration of 60 ng &#956;L<SUP>-1</SUP> 
  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&#146;s tissues 
  including kidneys and livers carry <I>EGFP</I> 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.}
    }