| SOX2, as an important transcription factor, has multiple functions in stem cell maintenance and early-stage differentiation of embryonic stem cells, the induction of IPS and activation or repression of many other genes. Meanwhile, the abnormal expression of SOX2 was proven to be related with many diseases and tumorigenesis.Many related genes and small molecule drugs which could affect the expression of SOX2 could be discovered by the study of the regulational expression of SOX2 gene. The discovery of these related genes and small molecule drugs would further clarify the multiple functions and greatly promote the application research of SOX2 gene. The main methods to detect the gene expression could be achieved by RT-PCR or the construction of a reporter gene expression vector under the control of the target promoter. The process of RT-PCR is complicated and not suitable for the high-throughput detection. On the other hand, the detecting method by the reporter gene expression vector under the control of the target promoter couldn't truly reflect the expression of target gene because of the occurrence of the epigenetic modification of endogenous promoter present in the genome.The emergence of artificial endonucleases technology makes the precious genome editing possible. There are three kinds of most widely used artificial endonucleases, including zinc finger nucleases (ZFN), transcription activator-like effector nuclease (TALEN) and CRISPR/Cas system by now. The recognition mechanism, construction methods and specificity of them are all different with each other. ZFN is the earliest artificial endonuclease, however, it has many disadvantages, for example, complicated process of ZFN assembling, the low successful rate of screening and off-target effects. These disadvantages limited the application of ZFN. Compared with ZFN, TALEN is easier to be assembled and has much higher specificity. In addition, CRISPR/Cas system is convenient for construction and has a short experiment period, but it has some off-target risk because its s recognition for target gene is based on base pair interaction between sgRNA with a short sequence and target gene.In this study, the establishment of two different Luciferase knock-in cell lines, SOX2P-Luciferase and SOX2P-SOX2-T2A-Luciferase, and their transcriptional regulation in vitro were investigated by using TALEN and CRISPR/Cas system. The research project can be divided into two parts as a whole. On one hand, two Luciferase knock-in reporter systems were established in HEK293 cells by placing a Luciferase gene at different loci in the genome under the control of the SOX2 promoter using TALEN technique. In this way we could build two cell lines for studying the regulation of SOX2 gene. On the other hand, to prove the reliability and sensitivity of this novel Luciferase knock-in system, an efficient transcriptional activation system was established using CRISPR/Cas technique and applied to the two knock-in cell lines to prove whether the Luciferase activity was directly correlated with the activity of SOX2 endogenous promoter. This novel system provides a useful tool for studying the SOX2 associated signal pathway, transcriptional regulation and tumor therapy research, and has great potential in medical and industrial application. The specific research contents of the project were as follows:(1) Designing, assembling and activity detection of TALENs targeting SOX2 gene. In order to use endogenous SOX2 promoter to control the Luciferase reporter gene, two strategies for genome editing were designed. One strategy was to insert a Luciferase gene right after the downstream of SOX2 promoter. The inserted Luciferase gene will destroy the expression of SOX2 on the genome. Another strategy was to insert the Luciferase reporter gene at the end of SOX2 gene, which would result in producing the SOX2-T2A-Luciferase fusion protein. In this case, the expression of SOX2 gene was not destroyed, both SOX2 and Luciferase protein could be separately produced by the cleavage peptide T2A. The expression of the fusion protein was under the control of SOX2 promoter. To achieve the goal, four target loci for SOX2 TALENs were selected using ZiFiT software according to the information of SOX2 promoter and structure. Then TALEs were assembled according to the sequences of target loci respectively a. Finally complete TALENs expression vectors were constructed, then the biological activity of TALENs were assayed by transfecting the vectors into the HEK293 followed by T7E1 assay method. The TALENs with a better biological activity will be applied to the experiments based on two strategies above-mentioned.(2) Construction of donor vectors for targeting SOX2 gene. In order to insert a Luciferase gene in the genome under the control of endogenous SOX2 promoter, we designed two different targeting vectors according to the location of selected TALENs with high cleavage efficiency. The first donor vector is to insert a Luciferase reporter gene right after endogenous SOX2 promoter. For this strategy, we designed an up homologous arm located upstream of the translation initiation site of SOX2 gene and a down homologous arm located downstream of the translation termination site. A Luciferase reporter gene, followed by a CMV-eGFP-T2A-Neomycin-SV40pA expression cassette was inserted between the up and down homologous arms. The fusion gene of PGK-TK-T2A-mCherry-SV40pA was placed outside the down homologous arm as a negative selection element to eliminate the randomly inserted cells. For the construction of second donor vector targeting at the end of SOX2 gene, we designed an up homologous arm ends right before the stop codon of SOX2 gene followed with T2A-Luciferase gene. The backbone of this vector is the same with the first donor vector above. The two donor vectors will be used to establish two corresponding cell lines.(3) The establishment of SOX2P-Luciferase and SOX2P-SOX2-T2A-Luciferase cell lines. The two selected TALENs were co-transfected with its corresponding donor vector into HEK293 cell lines, respectively. Then HEK293 cells were screened using G418 drug followed by GCV drug. The primary positive clones were obtained by diluted cloning followed by PCR analysis. Finally, the SOX2 Promoter-Luciferase and SOX2P-Luciferase and SOX2P-SOX2-T2A-Luciferase cell lines were obtained by further confirmation of sequencing and Southern Blot.(4) Designing and construction of sgRNA expression vector expressing sgRNAs targeting SOX2 promoter region. Seven sgRNAs were selected by searching SOX2 promoter sequence in NCBI and select target loci according to the design principle of sgRNA. The primer pairs for each sgRNA were designed and synthesized, then annealed at room temperate, which were ligated with sgRNA expressing vector. SOX2 sgRNA 1-7 expression vectors were obtained after identification of enzyme digestion and sequencing.These sgRNAs located at SOX2 promoter region will be used in activating SOX2 gene.(5) Establishment of CRISPR/Cas mediated transcriptional activation system. At first, the three transactivation related genes VP64, P65 and HSF1 were cloned respectively by PCR. Then dCas9-VP64 fusion protein expression vector and MS2-P65-HSF1 fusion protein expression vector were constructed, respectively. Finally sgRNA2.0 expression vector was generated by inserting a RNA aptamer sequence into the loop region of the vector backbone. MS2 protein could bind to the aptamer region specifically. The CRISPR/Cas mediated transcriptional activation system was developed by the combination of the expressing vectors carrying transactivation related genes and sgRNA expression vectors. Then this system was applied to the assay of transcriptional activation of SOX2 gene. The comparison of transcriptional activation effect of single sgRNA with that of multiple sgRNAs was also investigated based on CRISPR/Cas mediated transcriptional activation system.(6) Verifying whether the expression variation of Luciferase gene in SOX2P-Luciferase and SOX2P-SOX2-T2A-Luciferase cell lines could reflect the expression variation of endogenous SOX2 exactly. CRISPR/Cas mediated transcriptional activation system was applied to SOX2P-Luciferase, SOX2P-SOX2-T2A-Luciferase and HEK.293 cell line separately to verify whether the expression variation of Luciferase can reflect the expression variation of SOX2 gene accurately in the HEK 293 cells by the comparison of the result from experimental group and that from the control group.In conclusion, in this study TALENs was the first time used to insert a Luciferase reporter gene in situ at the downstream of SOX2 promoter or 3 terminal of SOX2 gene, respectively, which resulted in the establishment of two Luciferase knock-in HEK 293 cells, as SOX2P-Luciferase and SOX2P-SOX2-T2A-Luciferase cell lines. The foreign genes of two knock-in cell lines were proved to be exactly integrated in the genome from several aspects. In addition, CRISPR/Cas mediated transcriptional activation system was established and applied in SOX2P-Luciferase and SOX2P-SOX2-T2A-Luciferase cell lines. The result indicated that the expression level of Luciferase of the cell lines can reflect the expression of SOX2 gene of the cell lines accurately and sensitively. The two cell lines will be useful tools for the functional study of SOX2 and the screening of small molecules which could affect the expression of SOX2. At the same time, the methodology of this study can be exploited to construct any other reporter systems for the functional study of other genes. |
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