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Study On Exogenous Gene Silencing Of Transgenic Arbas Cashmere Goats

Posted on:2017-02-14Degree:DoctorType:Dissertation
Country:ChinaCandidate:M T NuoFull Text:PDF
GTID:1220330485966607Subject:Zoology
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Transgene silencing is a widespread phenomenon in transgenic plants and animals, bringing new problems for the cultivation and application of genetically modified organisms. Currently, the researches on the mechanism of transgene silencing have been extensively studied in the field. Based on previous reports, the transgene silencing phenomenon is thought to be associated with the methylation status of the exogenous gene promoters, the histone modification abnormalities and the copy number of exogenous gene. In the past 5 years, our research group obtained hundreds of transgenic cashmere goats which were transfected by DsRed (Reporter gene), and the phenomenon of transgene silencing was observed in the goats.In this research 6 representatives of the above goats were chosen for detection, and their ear tips fibroblasts and a part of the epidermal cells were isolated, bservation of the individual and the samples of the adult transgenic cashmere goats, the red fluorescence was not displayed in most of the individuals under the fluorescence excitation in the whole body level and cell level. It could be concluded that the red fluorescent protein was not expressed at the cell level. This result was not consistent with characteristics of the CMV promoter, and indicated that there was a phenomenon of transgenic silencing.After the detection experiments of genetically modified cashmere goats, we found that the expression cassette of exogenous DsRed gene in transgenic cashmere goats was unbroken and had complete function of gene expressing. After the real-time qPCR on the transgenic cashmere goats on the mRNA level, we found that the expression of DsRed mRNA was slightly higher than other individuals only in ZK110321, which was still lower than the expression level of nuclear donor cell CFC3-KI. The Western Blotting detection found that only ZK110324-1 individual and CFC3-KI cell could be detected the expression of DsRed protein in protien level. It could be concluded that CMV promoters in different tissues were silenced in different levels according to the difference expression of DsRed mRNA from fibroblasts and epidermal cells in ZK0311-1 and ZK110324-1. The exogenous gene copy numbers of the 6 transgenic cashmere goats were achieved by absolute quantification method of RT-qPCR, but there was no significant correlation with DsRed expression, excluding the effect of copy number of exogenous gene on the expression of DsRed gene.The methylation status of CMV promoters in transgenic Cashmere Goats was detected through Bisulfite Sequencing-PCR. Methylation status of CMV promoters from 6 healthy transgenic goats Fibroblasts and the non-cloned nuclear donor cell CFC3-KI had been tested. The results showed that CMV promoters in 4 adult transgenic goats were highly methylated (77.4%-88.2%),1 adult transgenic goat’s CMV promoter was in medium high methylation status (58.7%), only 1 adult transgenic goat CMV promoter was in a low methylation status (21%), and the methylation level of CMV promoter of non-cloned nuclear donor cell was very low (14.3%). Through association analysis of the above data and the expression of DsRed mRNA, it is found that the expression level of exogenous gene in transgenic cashmere goats was significantly negative correlated with the methylation status of its promoter. Those results indicated that main reason of the silencing of the exogenous gene was the high methylation level of exogenous CMV promoter.In order to re-express the exogenous gene in transgenic cashmere goats that were silenced, DNA methyltransferase inhibitor 5-Azacytidine (5-Az) and histone deacetylase inhibitor Trichostatin A (TSA) were used to treat the cells of transgenic cashmere goats in the experiment. At first, the fibroblasts and epidermal cells of ZK0311-1 and ZK110324-1 were screened to confirm optimal concentration. The results showed that the optimal 5-Az concentration was 5μmol/L, and TSA concentration was lμmol/L for fibroblasts treating; the optimal 5-Az concentration was 20μmol/L, TSA concentration was 1μmol/L for epidermal cells treating. The treating time of 5-Az was 7 days, and TSA was 24 hours. Then good results of the re-expression of DsRed exogenous were achieved through treated with this concentration on the ear tip fibroblasts. The expression of DsRed mRNA in fibroblasts obtained from ear tips of ZK110324-2 was increased 50.19 times through treated by 5-Az and 15.08 times through treated by TSA. The results of the methylation level of exogenous CMV promoter in 6 transgenic cashmere goats fibroblasts obtained from ear tips and nuclear donor cells treated by 5-Az showed that CMV promoter methylation levels were significantly decreased on the fibroblasts of ZK0311-1、ZK110324-1 and ZK110324-2, indicated the effectiveness of 5-Az decreases the exogenous CMV promoter methylation level. In addition, it was found that DsRed gene could re-express in exogenous silencing individual by only TSA treating, and the expression level of DsRed mRNA and TSA concentration showed the characteristics of dose-dependent growth, concluding that histone acetylation abnormality was one of the reasons of exogenous gene silencing in the transgenic cashmere goats.The fibroblasts of the ear-tip from the two transgenic cashmere goats, ZK0310-1 and ZK0311-1, which red fluorescent protein was not expressed at the whole body and cell level, were used for clone. After oocyte’s comprehensive reprogramming, the re-expression of red fluorescence protein in the nuclear transfered embryos was observed.Through the above experiment, it could be concluded that the expression of exogenous gene in different individual of transgenic cashmere goats was different, even was silenced, which was related to the methylation of CMV promoter and the abnormal acetylation of histones. Furthermore, the silencing levels of CMV promoter was different in diffrent tissues in transgenic cashmere goats; the expression of exogenous in transgenic cashmere goats could be increased by 5-Az and TSA treatment; The procedure that the cells of transgenic cashmere goats with silencing exogenous gene were used as donor cells for cloning might also contribute to the re-expression of the exogenous gene.
Keywords/Search Tags:Transgenic cashmere goat, Gene silencing, Promoter methylation, Histone acetylation, 5-Az, TSA, Somatic cell cloning
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