| With the development of economic and science research, it is often critical to make transgenic animals that express multiple genes under the control of a single promoter in biomedical research and agriculture. Now, different approaches, such as internal ribosomal entry site (IRES) elements, bidirectional or double promoters, or coinfection with multiple viral vectors are commonly used. These strategies, however, mostly suffer from the fundamental problem that coexpression of the heterologous proteins is unreliable and far from quantitative. In the case of IRES-mediated coexpression, for example, expression of the downstream cistron is sensitive to its specific positioning after the IRES and will not be come true; the upstream cistron in these bicistronic mRNA open reading frames (ORFs) is more strongly transcribed than that of downstream protein.2A peptide could improve the above defectiveness.2A peptide, known as cis-acting hydrolase element’(CHYSEL), could mediates expression of multi-gene indepently. Compared to IRES,2A peptide has a shorter sequence (54-90bp), genes in the2A based multi-gene vector could express more independently and efficiently, which owns the advantages that others couldn’t gain over it.At present, most of the production of transgenic animals are mediated by single-cistron vector, and animals mediated by multi-cistron vector are rare. hi2A peptide mediated transgenic animals, except mice and pigs, however, there is no report in sheep. Now, lentivirus vector based transgenic technology owns the highest efficiency in transgenic livestock production. Combining the lentivirus vector with2A-mediated polycistronic co-expression, the transgene efficiency would be high and multiple genes expressed coordinately. In one animal that express multiple exogenous gene at the same time, it is feasible to make many genes synergy or polymerization, which could improve the transgene utilization efficiency, shorten the production cost and animal production cycle. In the study, we make the fluorescent reporter gene (tdTomato, zsYellowl and acGFPl) as target gene, to facilitate testing the gene expression mediated by2A peptide. Firstly, tdTomato, zsYellowl and acGFPl were linked by2A peptide, subcloned and constructed the multi-gene vector pLEX-2A-TYG. Second, the lentiviral vector was transfected in to the293T and CHO cells, and the expression of the vector was investigated under confocal microscope. Upon this, the first tri-gene transgenic sheep were produced by injecting the lentiviral particles into the perivitelline space of sheep zygotes. Several methods such as PCR, Western blotting, Southern blotting, Real Time RT-PCR were used to analyze the transgene integration, copy numbers, transgene expression for trangene lambs. To analyze the transgene silencing, methylation level of the promoter and coding region in the vector was examined by Bisulfite sequencing PCR, BSP. Isolated fibroblasts from transgene lambs were treated by DNMT inhibitor5-azacytidine (5-azaC) and deacetylase inhibitor TSA, the DNA methylation mechanism was explored. Main points in the study were listed in the following:1We first constructed the fragment2A-linker with the size of102bp.2A-linker mediated tri-gene lentiviral pLEX-tdTomato-zsYellow1-acGFP1(pLEX-2A-TYG) was constructed successfully PCR, enzyme digestion and clone reaction.2The Tri-gene vector was transfected with the293T and CHO cells, which were detected by inverted fluorescent microscope and western blotting. Results showed that fluorescent proteins genes tdTomato, zsYellowl and acGFPl contained in the tri-gene vector could express high efficiently and independently.3Lentiviral particles were produced by cotransfection pLEX-2A-TYG vector with pMD2.G and packaging plasmid pSPAX2into293T cells using lipofectamine2000, with a high titre (3×109IU/ml). The lentivirals were used to infect293T and CHO cells,2A peptide mediated tdTomato, zsYellowl and acGFPl expressed efficiently and independently, which were detected by confocal microscope.4To test whether the multi-gene vector work well in vivo, transgenic sheep was produced by injecting the lentiviral particles into the perivitelline space of sheep zygotes. There are5donors and37recipient Merino sheep for use.37transgenic embryos were injected lentiviral particles, and transferred to37recipient rams. Of seven lambs born, two (#6and#7) of which were identified with exogenic gene by PCR.5Lambs were all examined by portable UV light, no fluorescent was found in the skin of the ears, heads, lips, feet and other bodies that can be seen. The skin of the transgenic lambs were gained and used as sample, we analyzed transgene expression by western blotting and RT-PCR. Unfortunately, no signal was detected.6To test the cause of transgene silencing, the methylation level of genome DNA in the tails of transgenic lambs were analyzed by BSP. Results showed that methylation levels in the CMV were76.25%(#6) and64.70%(#7); in the coding region of tri-gene construct, methylation levels were for lamb#6, tdTomato97.5%, zsYellow198.1%and acGFP199.2%; for lamb#7, tdTomato, zsYellowl and acGFPl were97.6%,99.5%and97.6%, respectively. It denoted that the hypermethylation existed in the tails of transgenic sheep. We implied that the silencing of transgene expression would caused by DNA methylation in the transgenic sheep.7To test whether DNA methylation played a critical role in the transgene expression, skins of two transgenic and one non-transgenic lamb were cut, the fibroblasts of which were isolated and purified. The cells were treated with5-azaC and TSA with different concentration and time, respectively. The results detected by inverted fluorescent microscope and FACS showed that number of the fluorescent (GFP+) cells increased with the drug concentration and incubation time increasing. When5-azaC and TSA co-incubated in the cells, GFP+cells were more than that treated with5-azaC or TSA alone, which suggested that there is a co-operation between5-azaC and TSA. Results above showed that expression of the fluorescent protein gene has an obvious negative relationship with DNA methylation and histone deacetylation;5-azaC and TSA had a coordinated effect when they work together.In conclusion, we successfully produced multi-gene sheep by2A peptide mediated tri-gene vector, which would provide new materials for transgenic research. Unfortunately, reporter genes of the tri-gene mediated by2A peptide expressed successfully in the cells, but silenced in the tails of the transgenic sheep. Expression of the reporter gene in part of fibroblasts isolated from transgenic sheep was reactivated when they were treated by DNMT inhibitor and deacetylase inhibitor, which denotes that DNA methylation plays an important role in transgene expression. The mechanism of transgene regulation, down-regulation of DNA methylation and methods of avoiding silencing of transgene would be studied in the future. |
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