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Dara, Mahintaj, Dianatpour, Mehdi, Razban, Vahid. (1398). Synthesis a New Viral Base Vector Carrying Single Guide RNA (sgRNA) and Green Florescent Protein (GFP). , 30(3), 205-209. doi: 10.22059/jsciences.2019.265567.1007316
Mahintaj Dara; Mehdi Dianatpour; Vahid Razban. "Synthesis a New Viral Base Vector Carrying Single Guide RNA (sgRNA) and Green Florescent Protein (GFP)". , 30, 3, 1398, 205-209. doi: 10.22059/jsciences.2019.265567.1007316
Dara, Mahintaj, Dianatpour, Mehdi, Razban, Vahid. (1398). 'Synthesis a New Viral Base Vector Carrying Single Guide RNA (sgRNA) and Green Florescent Protein (GFP)', , 30(3), pp. 205-209. doi: 10.22059/jsciences.2019.265567.1007316
Dara, Mahintaj, Dianatpour, Mehdi, Razban, Vahid. Synthesis a New Viral Base Vector Carrying Single Guide RNA (sgRNA) and Green Florescent Protein (GFP). , 1398; 30(3): 205-209. doi: 10.22059/jsciences.2019.265567.1007316

Synthesis a New Viral Base Vector Carrying Single Guide RNA (sgRNA) and Green Florescent Protein (GFP)

Journal of Sciences, Islamic Republic of Iran
مقاله 1، دوره 30، شماره 3، مهر 2019، صفحه 205-209    اصل مقاله (253.55 K)
نوع مقاله: Original Paper
شناسه دیجیتال (DOI): 10.22059/jsciences.2019.265567.1007316
نویسندگان
Mahintaj Dara1؛ Mehdi Dianatpour* 2؛ Vahid Razban1
1Department of molecular medicine, school of advanced medical science and technology, Shiraz University of medical science, Shiraz, Iran
2Genetic Shiraz university of medical sciences
چکیده
CRISPR/Cas9 system is a powerful gene editing tool in vivo and in vitro. Currently, CRISPR/Cas9 delivery cells or tissue with different vehicles are available, and Adeno- associated virus (AAV) in one of them. Due to AAV packaging size limitation, AAV base vectors that carry CRISPR/Cas9 system do not have florescent tag like GFP for simple detection and navigation of cells, containing AAV. The aim of this study was to modify and synthesis AAV base vector for CRISPR/cas9 system containing sgRNA and GFP.Px602 plasmid was double digested with NcoI and HindIII restriction enzyme. Gfp gene was amplified from px458 plasmid. Linear digested px602 and amplified Gfp gene were ligated together. After transformation and colony PCR on white colonies, plasmid was extracted and transfected to HEK-293 cell line. Gfp expression was monitored by florescent microscopy. After transfection of modified plasmid, florescent microscopy of HEK-293 cells showed shining green florescent cells, which indicate that Gfp gene, was replaced in the correct place according to our design.We modified an AAV base vector carrying CRISPR/Cas9 system, and synthesized a new vector carrying Gfp gene and sgRNA that can be packaged as reporter AAV for navigation and detection of cells, containing AAV.
کلیدواژه‌ها
CRISPR/Cas؛ AAV base vector؛ gene editing
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