Assembly Of A Positive Feedback Loop For T7rna Polymerase

Phage T7 RNA polymerase consists of a single polypeptide chain with a molecular weight of approximately 100 KD. The coupled expression system of T7 RNA polymerase has been used extensively for in vitro RNA transcription and in vivo gene expression in E. coli as well as in eukaryotic cells. The advantages of this coupled expression system include probably the followings: T7 RNA polymerase is the most efficient RNA polymerase found so far; the unique T7 promoter is strong and constitutively active; the system is not competitive for limited amount of transcription factors with the endogenous RNA polymerases. However, the expression of T7 RNA polymerase usually depends on the vaccinia vector, leading to inevitable contamination of viruses in the preparations. In this study, we assemble a positive feedback loop for the production of T7 RNA polymerase using basic molecular elements, including CMV promoter, T7 promoter and the internal ribosomal entry site (IRES). In order to verify this positive feedback system, two reporter constructs, the green fluorescence protein and firefly luciferase, driven by T7 promoter were constructed. The expression of EGFP and luciferase were quantitatively determined by real time RT-PCR, confocal microscopy, flow cytometry and dual luciferase activity assay. Application of this positive feedback loop for T7 RNA polymerase to the preparation of lentiviral vectors was reported in another study.Methods and Contents1. Construction of an expression vector for T7 RNA polymerase (T7RNP) driven by the CMV promoter. This expression vector was constructed using pEGFP-N1. To achieve this, T7RNP was first cloned by PCR, and ligated with pGM-T. The sequence was verified before T7RNP was subcloned behind the CMV promoter in pEGFP-N1. The correct orientation of T7RNP in the construct was verified with enzyme digestion. The vector was designated as pCMV_T7RNP.2. Construction of an expression vector for T7RNP and EGFP fusion protein driven by the CMV promoter. In order to delete the stop codon between T7RNP and EFGP in pCMV_T7RNP, the vector was cut with SmaI and KpnI. The plasmid was religated after the cohesive ends were blunt ended. The correct reading frame for the fusion protein was verified.3. Quantitative analysis of T7RNP transcription levels. 293FT cells were transfected with pCMV_T7RNP. Specific primers and probe were synthesized for the measurement of T7RNP using real time RT-PCR. The transfection time and DNA doses were determined to evaluate the optimal conditions for nuclear T7RNP expression.4. Construction of an expression vector for T7RNP driven by the T7 promoter. To achieve this, IRES and T7RNP was inserted into the multiple cloning site of pET28a. The orientation was verified by sequencing. The vector was designated as pT7_IRES_T7RNP.5. Determination of T7RNP transcript levels generated by the positive feedback loop. pCMV_T7RNP alone or in combination with pT7_T7RNP or pT7_IRES_T7RNP will be transfected into 293FT. The expression levels of T7RNP will be determined by real time RT-PCR. The establishment of this positive feedback loop depends on the presence of IRES. Therefore, pT7_T7RNP will be used as a negative control.6. Construction of a reporter construct for EGFP to measure the protein levels generated by the positive feedback loop. The CMV promoter or T7 promoter was inserted into the multiple cloning site of pGL3-Basic. After that, EGFP was cloned into pGL3-Basic behind the CMV promoter, while IRES and EGFP were put behind the T7 promoter. The vectors were designated as pCMV_EGFP and pT7_IRES_EGFP respectively. The expression of EGFP in 293FT cells will be measured by flow cytometry and confocal microscopy.7. Construction of a reporter construct for luciferase to measure the enzyme activity generated by the positive feedback loop. The CMV promoter or T7 promoter was inserted into the multiple cloning site of pGL3-Basic. After that, Luc was cloned into pGL3_Basic behind the CMV promoter, while IRES and Luc were put behind the T7 promoter. The vectors were designated as pCMV_Luc and pT7_IRES_Luc respectively.The expression of Luc in 293FT cells will be measured by dual luciferase activity assay. Measurement of the titers of lentiviral vectors elevated by the positive feedback loop.The transfer vector, packaging vector and envelop vector were constructed in another study, and designated as pT7_Lenti_EGFP, pT7_IRES_Gag/Pol, and pT7_IRES_VSVG respectively. The RNA titers, DNA titers and the transduction units will be measured in the presence of positive feedback loop. The regular lentiviral system, pRSV_Lenti_EGFP, will be used a control.Results:1. By using PCR, the T7RNP gene was cloned successfully from E. coli BL21 (DE3), and inserted it into the pGM_T vector. DNA sequencing showed that no mutation occurred in our PCR product. A HindⅢfragment from pGM-T was then inserted into pEGFP_N1. The orientation was verified by enzyme digestion. The vector was designated as pCMV_T7RNP.2. The vector for the fusion protein of T7RNP and EGFP was successfully constructed. The cytoplasmic location of T7RNP was confirmed when pCMV_T7RNP/EGFP was transfected into 293FT cells for 24 hrs. The picture was taken under a fluorescence microscope.3. Different doses of highly purified pCMV_T7RNP DNA were transfected into 293FT cells. The expression levels of T7RNP were measured by real-time PCR after 24-96 hr transfection.4. The IRES and T7RNP were successfully cloned into pET28a. The correct orientation was verified by restriction enzyme digestion. The vector was designated as pT7_IRES_T7RNP.5. The reporter expression vectors of EGFP driven by the CMV promoter and the T7 promoter were successfully constructed. The vectors were designated as pCMV_EGFP and pT7_IRES_EGFP respectively.6. The reporter expression vectors of luciferase driven by the CMV promoter and the T7 promoter were successfully constructed. The vectors were designated as pCMV_Luc and pT7_IRES_Luc respectively.Conclusions A series of expression vectors were constructed using basic molecular elements for the expression of T7RNP. The generation and the effects of the positive feedback loop of T7RNP will be determined in the eukaryotic 239FT cells. This T7RNP expression system will be valuable for the preparation of lentiviral vectors in the future.