ساخت مدل سلولی آلوده به ویروس HTLV-1 و مختل کردن ژن gag توسط سیستم CRISPR/Cas9
کد: G-1208
نویسندگان: مهرناز کفاشیان ℗, سعید عامل جامه دار ©
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دانلود: دانلود پوستر
خلاصه مقاله:
خلاصه مقاله
Background:Human T-cell leukemia virus type 1 is a lentivirus that belongs to the retroviridae family.The Khorasan province was reported to be as one of the endemic regions. Infection with HTLV-1 can lead to asymptomatic carrier state or two diseases including ATLL and HAM/TSP.The retroviral Gag protein is the critical structural protein that orchestrates particle assembly, release, and maturation to create an infectious virus.Interference with the structure of the gag leads to disruption of the assembly of the virus, which can be an attractive antiviral target for treatment.HTLV-1 currently cannot be treated with antiviral drugs.The recent advent of genome-editing technologies has enabled a new approach. In this study, we used CRISPR/ Cas9 technology to manipulate the gag gene to disrupt this important gene in HTLV-1 virus assembly.Method:In this study, the HEK 293T cell line was used to construct an HTLV-1 gag-infected cell . For this purpose, Gag gene from genomic DNA of MT-2 cells was amplified and cloned into transposon vector and transfected to HEK 293T.The presence of the transposon vector containing gag gene in the HEK 293Tcell line was confirmed by Fluorescent microscope, PCR, digestion with restriction enzyme and sequencing. Function of gag gene was evaluated by RT-PCR.In the next step, we designed sgRNA against Gag gene and cloned into Cas9 vector.This vector with second vector containing DsRed and Poly A,transfected to cellular model.In this study, gag gene disruption occurred with HITI-based CRISPR/Cas9 method.Results:The correct integration of the gag gene into the cell genome was confirmed by fluorescent microscope.The correct size of the PCR band was found to confirm the presence gag gene. In addition, the sequencing results showed that transfection was performed correctly.The disruption of gag was confirmed by fluorescent microscope which showed that the vector is inserted in the cell model.PCR was also performed to evaluate the correct integration of the second vector at the beginning of the gag gene. Finally,the sequencing results also showed that the knock in was done correctly.Conclusion:finally, we succeeded in using the CRISPR/Cas9 technique to destroy the HTLV-1 virus gag gene structure, which plays a key role in the assembly of HTLV-1 virus.
کلمات کلیدی
Keywords: HTLV-1, gag gene, Transposon system, CRISPR/Cas9, HITI
