Study On Gibson Assembly Based Novel Molecular Cloning Systems Construction And Application And SV40T Reprogramming Role In Immortalized Melanoblasts

This research contains three projects of novel molecular cloning system constructions, two melanoma gene therapy studies and one melanoma drug resistance gene screening subject. The after three are also confirmations and applications of the first three items. In addition, based on our previous study on melanoblast reversible immortalization, we investigated the SV40T reprogramming role in immortalized melanoblasts. Each part of the my whole research is relatively independent integrity and interrelated with each other.Gibson DNA assembly is developed by Daniel Gibson. It is a double stranded DNA molecules ligation method based on homologous sequences, has its unique advantage compared with the conventional molecular cloning ligation technique via restrict digestion enzyme sites. In this study, utilizing Gibson DNA Assembly, breaking a variety of limitation in previous molecular cloning technology, three kinds of molecular cloning technology innovation were invented:First, no recombination adenovirus construction system; second, multi-siRNA one step express system; third, a low cost, whole genome coverage, simple, random unbiased siRNA library.Our group has been investigating in the fields of stem cell biology and tumor biology for many years. Technology was developed for application, we tried those techniques we established in melanoma gene therapy and drug resistance target gene screening study. On one hand to validate the technical innovation in the scientific research practice, on the other hand to find new theoretical evidence and method for research and clinical practice of melanoma gene therapy and drug resistance gene screening.In addition, a project about SV40T reprogramming role in immortalized melanoblasts is also administrated in my research. This study is based on the melanoblast reversible immortalization issue in our lab. From the phenomenon of imc cells’s teratoma like mass forming ability in vivo, we investigated the reprogramming phenomena and the underline mechanism. Therefore to clarify the feasibility and security of sv40-t antigen using to immortalize cell lines, provide more clues for the traditional SV40-T antigen immortalization technology theory.The main results and conclusions are as follows:1. We established a no shuttle adenovirus system, constructed vector plasmid pROS and pGOS. Used pROS for function test, did Gibson Assembly ligation with GFP as the target gene. Successfully bypassed the steps of shuttle plasmid and the recombinant. Cloned the target gene to the complete adenovirus backbone plasmid directely. The positive clone rate is more than 70%, the adenovirus plasmid can be successfully packaged and amplified. Cell that infected the virus can express green fluorescent protein efficiently. This new adenovirus construction method has high efficiency, simple process, low cost and avoids the high risk of mutation in the recombination step. This system is a superior adenovirus construction system.2. We constructed one step multi-siRNA expression vector plasmids:retrovirus (pSOK),adenovirus (pAdTrace-OK, pAdTrack-OK) and Piggy-bac (pBOK). Designed and constructed the pB2B plasmid that contain back to back H1/U6 sequence. This plasmid can be used to prepare inserts for multi-siRNA one step system. For function test, we used pSOK as vector to do beta-catenin interference tests and found that this method can ligate multiple interference fragment in one step reaction with short-term, low-cost and high efficiency, in mice and human cell lines, in vitro and in vivo experiments confirmed the multi-siRNA one step expression system can efficiently silence target gene. It is an excellent new gene silencing methods.3.We constructed a randomly whole genome coverage siRNA retrovirus library, using pSOK retroviral backbone plasmid as the vector, randomly chemical synthetic 19 bp siRNA sequence as inserts, Gibson DNA Assembly as ligation strategy, clone PCR, cloning counting, sequencing and nfected cells’heterogeneous observation results shows that one time synthesis of library can generate 2-3×106 clones with randomness and heterogeneity. After been infected by the retrovirus library, imc23 cells showed high infection efficiency and heterogeneity clones generation phenotype. The protocol is simple and time-saving. It is an excellent siRNA library construction technology, has wild research and clinical prospect.4. We used no shuttle adenovirus construction system designed IGF-1R domain negative protein expression adenovirus AdRdnIGF-1Rα and AdRdnIGF-1Rβ,both of them can inhibit the growth of human and mouse melanoma. AdRdnIGF-1Rβ is better than AdRdnIGF-1Rα. The main reason is promoting tumor apoptosis, inhibiting cells go into mitosis phase playing a partially role. This consequence told us that secreted IGF-1R domain negative protein can be a new method for gene therapy of melanoma. These data also proved that our no shuttle adenovirus system is highly efficient, easy practicable and can strongly and stably express target genes.5. Use multi-siRNA express adenovirus system, we constructed 3 targeting sites IGF-1R interference adenovirus for human (AdRhIGF1R-OK) and mice (AdRmIGF1R-OK). Use human melanoma cell line B16F10 and mouse melanoma cell line A375 as cell model, we demonstrated that the multi-siRNA adenovirus could be constructed efficiently and easily. AdRhIGF1R-OK and AdRmIGF1R-OK have strong interference effect, can obviously inhibit the growth of melanoma in vitro and in vivo. The main reason is promoting tumor apoptosis. AdRhIGF1R-OK could be used as a new means of gene therapy of melanoma.6. We infected human melanoma A375 cells with our siRNA library, then treated the cells by current first line chemotherapy drugs to get drug resistant cell clones. According to the traceable siRNA fragment in the drug resistant cell clones, we can find out the silenced gene which may relate to drug sensitivity and drug resistance. At present, we have successfully got vemurafenib resistant A375 cells. The drug resistance is strong and stable, the cells can survive when vemurafenib concentration at 100uM, while control cells die out at 25uM. Present results proved that our siRNA library is convenient, wide cover, random and has strong silence efficiency in host cells. The acquirement of drug resistant cell clones is a milestone in our project. On one hand, this step proved the feasibility of our experimental scheme, one the other hand, it is a strong foundation of the the following logical study. It is expected to provide new clues for melanoma clinical treatment, find new targeting genes relate to drug resistance and sensitivity.7. We discovered and confirmed that SV40-T immortalized melanoblast cells were reprogrammed to some extent by SV40-T. Compared with the primary melanoblast cells, SV40-T immortalized melanoblast cells were more primitive and had increased differentiation potential. The four IPS factors except Sox2 were increased, a number of pluripotential genes, neural crest specific genes were up regulated, sphere forming ability were evaluated. Melanocyte lineage differentiation related markers were unexpected up regulated, maybe that is why imcx kept strong melanocyte lineage proficiency. Sox2 was not required in SV40-T mediated melanoblast cell reprogramming, may even not be allowed to be at high expression level. Imcx cells highly expressed some specific markers of three germ layers and were demonstrated could terminally differentiate into melanoma cells, neurons, glial cells, bone tissue and adipose tissue. Masses formed in cell transplantation experiment exhibit characteristics like terotoma. Increase of E2f and decrease of p53 may relative to the reprogramming by SV40-T. In vivo transplantation, treated with different IPS factors,imcx teratoma forming situation were very different. C-Myc and SV40-T had a synergistic effect, and Sox2 had the opposite effect.In summary, this research made three improvements and innovations in adenovirus construction, RNA interference, siRNA library technology; found new methods and theoretical basis for melanoma gene therapy and drug resistance; first discovered the SV40-T reprogramming role in immortalized melanoblast cells, enriched the theory system of cell immortalization by SV40-T technique.