The Research On Methylation Of Exogenous Gene In Transgenic Sheep And Nanometer Transgenic Technology

With the development of transgenic technology, transgenic animal biological technology has beensuccessfully used in many aspects, but the exogenous gene in vivo, especially in large transgenic animalexpression is still difficult to predict. DNA methylation is an important epigenetic modification, regulatesmany biological processes in the body, occurs in the methylation of promoter region usually inhibit genetranscription and expression. This study aimed to investigate the methylaotion leves of exogenous genesand promoters and the difference of protein expression in transgenic sheep obtained by different transgenictechnologies. Bisulfite sequencing method was adopted to detect the methylation status of the promoterregion and coding region region exogenous in tail tissues of transgenic sheep, The results is that themethylation leve of promoter region with stem cell cloning was the highest, followed by somatic cellcloning，while that with perivitelline injection was the lowest. The methylation leves of the eGFP codingregion with perivitelline injection was the highest; followed by stem cloning; he methylation leves of theFGF5coding region with somatic cell cloning was higher than that was perivitelline injection. Westernblotwas adopted to detect the expression leve of exogenous protein, The results is that the exogenous proteinwas negatively correlated with methylaotion leve of the promoter region. This study indicate that thedifferent transgene method may affect the methylation leve of exogenous gene, thus affecting exogenousgene expression.The nanometer material can combined with target gene, and delivery the exogenous genes into the cells,and reprogramming to induce nuclear, thus preparation of transgenic animal. At first, cloned the eukaryoticexpression vector of transcription factor SOX2, which can induced pluripotent cells and involved in thenuclear reprogramming. And Then Preparation of molecular material with nano structure by using MgCl₂,AlCl3, NaOH three kinds of chemical substances, carrying the plasmid SOX2by transfection into cells, andexpression protein. The result is Mg₂Al-Cl-LDH nanoparticles had a width of about100nm，hexagonalstructure, Cell activity of nanometer concentration was200μ g/ml to87%, DNA and LDH mass ratio was4/50transfection rate reached11.17%, DNA combined with LDH time within15minutes the transfectionrate was6.93%, After cultured for24hours the transfection rate of17.4%training, The transfectionefficiency of C2C12cells better than HEK293T cells under the same conditions. Through the inspection canalso see Sox2expression plasmid enhanced green fluorescent protein was evident in cells, as result the Thestructural of plasmids is integrity, the sequence correct，and good expression ability.In conclusion, this study found that the level of methylation were negatively correlated with proteinexpression in transgenic animal, and suggesting that different transgenic method may influence theexpression of exogenous gene. Then establish and optimize the new method by nano material, provides anew train of thought and lay a new foundation for the development of transgenic animal technology.