Inheritance And Expression Stability Of Transgene In Transgenic Pigs

Transgenic animals have been used to study gene function, produce important proteins, xenotransplantation donor and generate models for the study of human diseases. Recent progress in animal cloning has provided an attractive alternative to improve transgenic efficiency, through the combination of transfection and somatic cell nuclear transfer (SCNT). Now many kinds of transgenic animals have been successfully produced by SCNT, such as mice, cattle, sheep and pig, and we also produced eGFP transgenic pigs by SCNT in 2008.However, the ultimate aim of transgenesis is not to produce several transgenic animals, but to service for the needs of human. In animal production, transgenic technology has been used to breed new livestock, and in our country, it is attracting a lot of attention, but it has been evidenced that inheritance and expression instability of transgene in transgenic animals is still the major limitation. Therefore, we checked transgene integration sites, copy numbers, DNA methylation and histone H3/H4 and H3K4/H4K16 acetyltion of CMV promoter in founder transgenic pigs and its offspring, to demonstrate the regulatory mechanisms of transgene by position effect, heredity effect, epigenetic modification and nuclear reprogramming.The results were as follows: 1.Porcine fetal fibroblasts treated with 0.5uM DNA methyltransferase inhibitor 5-Aza-dC for 48h or 0.25 uM histone deacetylase inhibitor TSA for 24h had no significantly relevant deaths and no considerably morphological changes;2. Silenced transgene could be reactivated with 5-Aza-dC or/and TSA treatment by reversing the CMV promoter status of histone hypoacetylation and DNA hypermethylation. Furthermore, the combination treatment once per 10 days could maitain transgene expression in a high level for more than 60 days by sustaining DNA hypomethylation and histone hyperacetylation;3.Significant differences in GFP expression were observed in various tissues of transgenic pigs (P<0.05), and in most tissues, except in ovary (p=0.063), the expression levels between K25-2 and K25-3 were significant difference (p<0.001);4.The differences in DNA methylation and acetylation of specific histone H3 and H4 lysines, including H3K9 acetylation, in various tissues of transgenic pigs were significant (p<0.001);5.After the combination treatment, DNA methylation and enrichment of histone acetylations at CMV promoter reached to a similar level without significant difference (P>0.05), and the difference of transgene expression level between cell lines declined, however, the expression difference was still significant (p<0.001);6.Transgene expression decreased in transgenic porcine fetal fibroblasts cultured over a long time course in vitro, with aging in transgenic pigs and during passage (p<0.001); 7. Methylation of CMV promoter increased in transgenic porcine fetal fibroblasts cultured over a long time course in vitro, with aging in transgenic pigs and during passage;8.Copy number of transgene decreased in transgenic porcine fetal fibroblasts cultured over a long time course in vitro, with aging in transgenic pigs and during passage (p<0.001);9. Varied transgene copies were observed in different tissues of transgenic pigs;10. The correlation of transgene expression level with copy number was positive (βC>0), and negative with promoter methylation(βM<0);...