fa ساخت و کلونینگ وکتور CRISPR/Cas9 هدفمند ژن CDKL5 و تایید هویت خط سلولی با روش QF-PCR علوم پایه Basic Sciences پژوهشي اصیل Original Atricle <span style="font-size:11pt"><span style="line-height:150%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">زمینه و هدف</span></span></span></b><b><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times="">:</span></span></b> <span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">اختلال طیف اوتیسم</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> (ASD) </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">و اختلال کمبود</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> CDKL5 </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">از جمله اختلالات عصبی-رشدی با ریشه ژنتیکی هستند که نیازمند مدل‌های دقیق مولکولی برای مطالعه مکانیسم‌های بیماری و توسعه درمان‌های نوین می‌باشند. سیستم ویرایش ژنوم</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> CRISPR/Cas9 </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">ابزاری کارآمد برای ایجاد تغییرات هدفمند در ژنوم است. هدف این مطالعه، طراحی و ساخت وکتور</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> CRISPR/Cas9 </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">حامل</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> sgRNA </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">اختصاصی برای ژن</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> CDKL5 </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">و تایید کلونینگ و هویت خط سلولی مورد استفاده به کمک روش‌های مولکولی بود</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times="">.</span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:150%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">روش بررسی</span></span></span></b><b><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times="">:</span></span></b> <span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">در این مطالعه تجربی، ابتدا</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> sgRNA </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">اختصاصی برای ناحیه مورد نظر در ژن</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> CDKL5 </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">طراحی شد. الیگوهای</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> Forward </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">و</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> Reverse </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">آنیل شده و در وکتور</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> PX458 </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">کلون شدند. پس از ترانسفورماسیون باکتری </span></span></span><i><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times="">E. coli</span></span></i> <span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">سویه</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> DH5&alpha;</span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">، کلونی‌های مثبت به روش</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> Colony PCR </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">و توالی‌یابی تایید شدند. همچنین جهت اطمینان از هویت خط سلولی</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> HEK293T </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">مورد استفاده، آزمون</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> QF-PCR </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">برای تعیین جنسیت و بررسی کروموزومی انجام شد</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times="">.</span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:150%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">یافته‌ها</span></span></span></b><b><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times="">:</span></span></b> <span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">نتایج</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> Colony PCR </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">وجود باند مورد انتظار 230 جفت بازی را در کلونی‌های منتخب نشان داد. توالی‌یابی نیز</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> Insert </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">صحیح</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> sgRNA </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">را در وکتور تایید کرد. نتایج</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> QF-PCR </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">الگوهای</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> Peaks </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">مشخص برای کروموزوم‌های جنسی را نشان داد که هویت خط سلولی را تایید می‌کرد</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times="">.</span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:150%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">نتیجه‌گیری</span></span></span></b><b><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times="">:</span></span></b> <span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">ساخت وکتور</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> CRISPR/Cas9 </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">هدفمند ژن</span></span></span><span dir="LTR" style="line-height:150%"><span new="" roman="" style="font-family:" times=""> CDKL5 </span></span><span lang="AR-SA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">با موفقیت انجام شد و تاییدیه‌های مولکولی لازم شامل کلونینگ و بررسی خط سلولی حاصل گردید. این وکتور می‌تواند برای مطالعات بعدی ویرایش ژن در مدل‌های سلولی مورد استفاده قرار گیرد</span></span></span><span lang="FA" style="font-size:12.0pt"><span style="line-height:150%"><span b="" nazanin="" style="font-family:">.</span></span></span></span></span></span></span></span><br> &nbsp; <span style="font-size:11pt"><span style="line-height:150%"><span sans-serif="" style="font-family:Calibri,"><b><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Autism Spectrum Disorder (ASD) and CDKL5 deficiency disorder are among the nervous and developmental disorder with genetic origin that requires accurate molecular models to study the mechanisms of disease and develop new treatments. The CRISPR / Cas9 genome editing system is an efficient tool to generate targeted changes in the genome. The aim of this study was to design and construct CRISPR / Cas9 vector carrier sgRNA for <i>CDKL5</i> gene and to confirm the cloning and identity of the cell line used by molecular methods</span></span></b><b><span dir="RTL" lang="FA" style="font-size:10.0pt"><span style="line-height:150%"><span arial="" style="font-family:"> .</span></span></span></b><b><span style="line-height:150%"><span style="font-family:"Times New Roman","serif""></span></span></b></span></span></span><br> <span style="font-size:11pt"><span style="line-height:150%"><span sans-serif="" style="font-family:Calibri,"><b><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Materials and methods: in this experimental study, at first, specific primers were designed for region in the cdkl5 gene </span></span></b><b><span dir="RTL" lang="FA" style="font-size:10.0pt"><span style="line-height:150%"><span arial="" style="font-family:">.</span></span></span></b><b><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Oligo nouclotide forward and reverse annealing and cloned in PX458 vector. After transformation of E. coli strains DH5, positive colonies were confirmed by PCR and sequencing methods. Also to ensure the identity of the HEK293T cell line, QF - PCR was used to determine the sex and chromosome study</span></span></b><b><span dir="RTL" lang="FA" style="font-size:10.0pt"><span style="line-height:150%"><span arial="" style="font-family:">.</span></span></span></b><b><span style="line-height:150%"><span style="font-family:"Times New Roman","serif""></span></span></b></span></span></span><br> <span style="font-size:11pt"><span style="line-height:150%"><span sans-serif="" style="font-family:Calibri,"><b><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Results: colony - PCR results showed the expected band of 230 bp in selected colonies. Sequencing also confirmed insert sgRNA in the vector. The results of QF-PCR demonstrated specific peaks patterns for sex chromosomes that confirmed cell line identity</span></span></b><b><span dir="RTL" lang="FA" style="font-size:10.0pt"><span style="line-height:150%"><span arial="" style="font-family:">.</span></span></span></b><b><span style="line-height:150%"><span style="font-family:"Times New Roman","serif""></span></span></b></span></span></span><br> <span style="font-size:11pt"><span style="line-height:150%"><span sans-serif="" style="font-family:Calibri,"><b><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Conclusion: the CRISPR / Cas9 target gene expression was successfully performed and molecular approvals were obtained for cloning and analysis of the cell line. This vector can be used for further studies of gene expression in cellular models.</span></span></b><b><span dir="RTL" lang="FA" style="font-size:10.0pt"><span style="line-height:150%"><span style="font-family:"Arial","sans-serif""></span></span></span></b></span></span></span><br> &nbsp; CRISPR/Cas9, ژن CDKL5, کلونینگ, QF-PCR, خط سلولی HEK293T CRISPR /Cas9 , cloning , QF - PCR, CDKL5 0 0 http://jams.arakmu.ac.ir/browse.php?a_code=A-10-3888-3&slc_lang=fa&sid=1 Fatemeh Faghihi فاطمه فقیهی yaldafaghihi@yahoo.com 4600319475328460092643 0903-1033-9555-0000 No PhD Student, Department of Biology, SR.C., Islamic Azad University, Tehran, Iran دانشجوی دکتری، گروه زیست شناسی،‌‌ واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران Jafar Amani جعفر امانی هارونی jafar.amani@gmail.com 4600319475328460092644 4600319475328460092644 Yes Professor, Applied Microbiology Research Center, Biomedicine Technologies Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran انستیتو فناوری‌های پزشکی، دانشگاه علوم پزشکی بقیه الله، تهران، ایران Milad Gholami میلاد غلامی dr.gholami@arakmu.ac.ir 4600319475328460092645 4600319475328460092645 No Associate Professor, Department of Biochemistry and Genetics, School of Medicine, Arak University of Medical Sciences, Arak, Iran گروه بیوشیمی و ژنتیک، دانشکده پزشکی، دانشگاه علوم پزشکی اراک، اراک، ایران Iman salahshourifar ایمان سلحشوری فر isalahshouri@iau.ir 4600319475328460092646 4600319475328460092646 No Assistant Professor, Department of Biology, SR.C., Islamic Azad University, Tehran, Iran گروه زیست‌شناسی،‌‌ واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران