fa ساخت و کلونینگ وکتور CRISPR/Cas9 هدفمند ژن CDKL5 و تأیید هویت خط سلولی با روش QF-PCR علوم پایه Basic Sciences پژوهشي اصیل Original Atricle <span style="font-size:11pt"><span style="line-height:101%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span calibri="" style="font-family:"><b><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black">مقدمه:</span></span></span></span></b> <span lang="FA" style="font-size:9.0pt"><span style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black">اختلال طیف اوتیسم </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:101%"><span new="" roman="" style="font-family:" times=""><span style="color:black">(Autism Spectrum Disorder) ASD</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black"> و اختلال کمبود </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:101%"><span new="" roman="" style="font-family:" times=""><span style="color:black">CDKL5</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black"> 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><span dir="LTR" style="font-size:9.0pt"><span style="line-height:101%"><span new="" roman="" style="font-family:" times=""><span style="color:black">CRISPR/Cas9</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black"> ابزاری کارآمد برای ایجاد تغییرات هدفمند در ژنوم است. هدف این مطالعه، طراحی و ساخت وکتور </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:101%"><span new="" roman="" style="font-family:" times=""><span style="color:black">CRISPR/Cas9</span></span></span></span><span style="font-size:9.0pt"><span style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black"> حامل </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:101%"><span new="" roman="" style="font-family:" times=""><span style="color:black">sgRNA</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black"> اختصاصی برای ژن </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:101%"><span new="" roman="" style="font-family:" times=""><span style="color:black">CDKL5</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black">و تأیید کلونینگ و هویت خط سلولی مورد استفاده به کمک روش‌های مولکولی بود</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black">. </span></span></span></span><span lang="FA" style="line-height:101%"><span b="" nazanin="" style="font-family:"><span style="color:black"></span></span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:104%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span calibri="" style="font-family:"><b><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black">روش کار:</span></span></span></span></b><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black"> در این مطالعه تجربی، ابتدا</span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black"> sgRNA </span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black">اختصاصی برای ناحیه مورد نظر در ژن</span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black"> CDKL5 </span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black">طراحی شد. الیگوهای</span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black"> Forward </span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black">و</span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black"> Reverse</span></span></span></span><span style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black"> آنیل شده و در وکتور</span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black"> PX458 </span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black">کلون شدند. پس از ترانسفورماسیون باکتری </span></span></span></span><i><span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black">E. coli</span></span></span></span></i> <span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black">سویه </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black">DH5&alpha;</span></span></span></span><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black">، کلونی‌های مثبت به روش</span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black"> Colony PCR</span></span></span></span><span style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black"> و توالی‌یابی تأیید شدند. همچنین جهت اطمینان از هویت خط سلولی </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black">HEK293T</span></span></span></span> <span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black">مورد استفاده، آزمون</span></span></span></span> <span dir="LTR" style="font-size:9.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black">QF-PCR</span></span></span></span> <span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:104%"><span b="" nazanin="" style="font-family:"><span style="color:black">برای تعیین جنسیت و بررسی کروموزومی انجام شد. </span></span></span></span><span dir="LTR" style="font-size:10.0pt"><span style="line-height:104%"><span new="" roman="" style="font-family:" times=""><span style="color:black"></span></span></span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:102%"><span style="direction:rtl"><span style="unicode-bidi:embed"><span calibri="" style="font-family:"><b><span lang="AR-SA" style="font-size:9.0pt"><span style="line-height:102%"><span b="" nazanin="" style="font-family:"><span style="color:black">یافته‌ها:</span></span></span></span></b> <span lang="FA" style="font-size:9.0pt"><span style="line-height:102%"><span b="" nazanin="" style="font-family:"><span style="color:black">نتایج </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:102%"><span new="" roman="" style="font-family:" times=""><span style="color:black">Colony PCR</span></span></span></span><span style="font-size:9.0pt"><span style="line-height:102%"><span b="" nazanin="" style="font-family:"><span style="color:black"> وجود باند مورد انتظار 230 جفت بازی را در کلونی‌های منتخب نشان داد. توالی‌یابی نیز </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:102%"><span new="" roman="" style="font-family:" times=""><span style="color:black">Insert</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:102%"><span b="" nazanin="" style="font-family:"><span style="color:black"> صحیح </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:102%"><span new="" roman="" style="font-family:" times=""><span style="color:black">sgRNA</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:102%"><span b="" nazanin="" style="font-family:"><span style="color:black"> را در وکتور تأیید کرد. نتایج </span></span></span></span><span dir="LTR" style="font-size:9.0pt"><span style="line-height:102%"><span new="" roman="" style="font-family:" times=""><span style="color:black">QF-PCR</span></span></span></span><span lang="FA" style="font-size:9.0pt"><span style="line-height:102%"><span b="" nazanin="" style="font-family:"><span style="color:black">الگوهای پیک‌های مشخص برای کروموزوم‌های جنسی را نشان داد که هویت خط سلولی را تأیید می‌کرد.</span></span></span></span> <span dir="LTR" style="font-size:10.0pt"><span style="line-height:102%"><span new="" roman="" style="font-family:" times=""><span style="color:black"></span></span></span></span></span></span></span></span></span><br> <b><span dir="RTL" lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span b="" nazanin="" style="font-family:"><span style="color:black"><span style="letter-spacing:-.1pt">نتیجه‌گیری:</span></span></span></span></span></b><span dir="RTL" lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span b="" nazanin="" style="font-family:"><span style="color:black"><span style="letter-spacing:-.1pt"> ساخت وکتور </span></span></span></span></span><span style="font-size:9.0pt"><span style="line-height:115%"><span new="" roman="" style="font-family:" times=""><span style="color:black"><span style="letter-spacing:-.1pt">&nbsp;CRISPR/Cas9</span></span></span></span></span><span dir="RTL" lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span b="" nazanin="" style="font-family:"><span style="color:black"><span style="letter-spacing:-.1pt">هدفمند ژن </span></span></span></span></span><span style="font-size:9.0pt"><span style="line-height:115%"><span new="" roman="" style="font-family:" times=""><span style="color:black"><span style="letter-spacing:-.1pt">&nbsp;CDKL5</span></span></span></span></span><span dir="RTL" lang="AR-SA" style="font-size:9.0pt"><span style="line-height:115%"><span b="" nazanin="" style="font-family:"><span style="color:black"><span style="letter-spacing:-.1pt">با موفقیت انجام شد و تأییدیه‌های 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></span><span dir="RTL" lang="FA" style="font-size:9.0pt"><span style="line-height:115%"><span b="" nazanin="" style="font-family:"><span style="color:black"><span style="letter-spacing:-.1pt">.</span></span></span></span></span> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:normal"><span calibri="" style="font-family:"><b><span style="font-size:8.0pt"><span ms="" style="font-family:" trebuchet="">Introduction:</span></span></b><span style="font-size:8.0pt"><span ms="" style="font-family:" trebuchet=""> Autism Spectrum Disorder (ASD) and CDKL5 deficiency disorder are among the nervous and developmental disorder with genetic origin that requires accurate molecular models are required 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 CDKL5 gene and to confirm the cloning and identity of the cell line used by molecular methods.</span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:normal"><span calibri="" style="font-family:"><b><span style="font-size:8.0pt"><span ms="" style="font-family:" trebuchet="">Methods:</span></span></b> <span lang="EN-GB" style="font-size:8.0pt"><span ms="" style="font-family:" trebuchet="">In this experimental study, at first, specific primers were designed for region in the cdkl5 gene. Oligo nouclotide forward and reverse annealing and cloned in PX458 vector. After transformation of <i>E. coli</i> 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><span style="font-size:8.0pt"><span ms="" style="font-family:" trebuchet="">.</span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:normal"><span calibri="" style="font-family:"><b><span style="font-size:8.0pt"><span ms="" style="font-family:" trebuchet="">Results: </span></span></b><span lang="EN-GB" style="font-size:8.0pt"><span ms="" style="font-family:" trebuchet="">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><span style="font-size:8.0pt"><span ms="" style="font-family:" trebuchet="">.</span></span></span></span></span></span></span><br> <b><span style="font-size:8.0pt"><span style="line-height:115%"><span ms="" style="font-family:" trebuchet=""><span style="letter-spacing:-.2pt">Conclusions:</span></span></span></span></b> <span lang="EN-GB" style="font-size:8.0pt"><span style="line-height:115%"><span ms="" style="font-family:" trebuchet=""><span style="letter-spacing:-.2pt">The CRISPR / Cas9 target gene expression was successfully performed and molecular approvals were obtained for cloning and analysis of the cell line. This vector is proposed for further studies on gene expression in cellular models</span></span></span></span><span style="font-size:8.0pt"><span style="line-height:115%"><span ms="" style="font-family:" trebuchet=""><span style="letter-spacing:-.2pt">.</span></span></span></span> CRISPR/Cas9؛ ژن CDKL5؛ کلونینگ؛ QF-PCR؛ خط سلولی HEK293T CRISPR /Cas9, Cloning, QF–PCR, CDKL5 19 25 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 4600319475328460093185 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 4600319475328460093186 4600319475328460093186 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 4600319475328460093187 4600319475328460093187 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 4600319475328460093188 4600319475328460093188 No Assistant Professor, Department of Biology, SR.C., Islamic Azad University, Tehran, Iran گروه زیست‌شناسی،‌‌ واحد علوم و تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران