افزايش ميزان بيان ژن Sox2 در سلول‌هاي فيبروبلاست گاوي با استفاده از TALE-TFs

Up-regulation of Sox2 Gene in Cattle Fibroblast Cells using TALE-TF

ژن Sox2 كد كننده یك فاكتور رونویسی موثر در ایجاد و پایداری خاصیت چند توانی سلول های بنیادین پستانداران می باشد. در این مطالعه توانایی Transcription Activator Like-Effector Transcription Factors (TALE-TFs) جهت افزایش بیان اندوژنوس ژن Sox2 در سلول‌های فیبروبلاست گاوی مورد بررسی قرار گرفت. به این منظور در ابتدا 320 جفت باز از منطقه پروموتری ژن Sox2 گاوی توالی یابی شد و در ادامه این توالی با اطلاعات ژنوم انسان موجود در بانك جهانی ژن مقایسه شد. هم‌ردیفی توالی های به دست آمده نشان داد كه این منطقه دارای مشابهت 44/93 درصد با منطقه پروموتری ژن Sox2 انسانی است. لذا با توجه به شباهت موجود توالی ها، از یك Sox2-TALE-TF كه قبلاً جهت افزایش بیان ژن Sox2 در سلول های انسانی استفاده شده بود برای تست فعالیت TALE-TF ها در سلول های فیبروبلاست گاوی استفاده گردید. جهت اندازه‌گیری میزان فعالیت TALE-TF ها در سلول های فیبروبلاست گاوی از آنالیز بیان ژن (qRT-PCR) و یك سیستم گزارشگر رنگ فلورسنت mCherry تحت كنترل پروموتر minCMV استفاده گردید. بررسی عكس های میكروسكوپ فلورسنت نشان داد كه پروموتر minCMV در سلول‌های فیبروبلاست گاوی به شكل كاملاً قوی عمل می كند و به عنوان یك گزارشگر برای فعالیت TALE-TF ها قابل استفاده نیست. آنالیز نتایج بیان ژن Sox2 گاوی با استفاده از تكنیك qRT-PCR نشان داد كه بیان ژن Sox2 در سلول های فیبروبلاست گاوی ترانسفكت شده با Sox2-TALE-TF به میزان 529/3 برابر افزایش داشته است. نتایج این تحقیق نشان داد كه TALE-TF ها به عنوان فاكتور های رونویسی مصنوعی می‌توانند جهت افزایش بیان اندوژنوس ژن ها در سلول های گاوی به كار برده شوند.

Introduction Cellular processes can be controlled by induction or repression of gene expression by introduction of exogenous genes and RNA interference (RNAi), respectively. However, the use of recently-developed specific DNA-binding transcription factors offer various advantages when compared to the exogenous gene introduction and RNAi for up or down regulation of gene expression. Sox2 gene encodes one of the most important transcription factors involved in the pluripotency state of mammalian cells. In this study the potential use of Transcription Activator-Like Effector-Transcription Factors (TALE-TFs) for endogenous up-regulation of Sox2 gene in cattle fibroblast cells were examined. Materials and Methods At first, a 320 base pair region of bovine Sox2 gene were sequenced and showed a 93.44% identity with its human orthologs. Based on the sequencing results, a previously reported Sox2-TALE-TF for human Sox2 gene was used for testing the functionality of TALE-TFs in cattle fibroblast cells. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 50 µg/ml Penicillin and Streptomycin (Invitrogen), 1% non-essential amino acids, and 10% fetal bovine serum (FBS). For detecting the Sox2-TALE-TF activity the qRT-PCR technique and a florescent based reporter system under the control of minCMV promoter were used. For quantitative RT-PCR analysis of Sox2 mRNA levels, the cells were lysed, and total RNA isolated by using the RNeasy kit (Qiagen Inc.) and subsequently cleaned up with Turbo DNase (Ambion) according to the manufacturer’s instructions. Reverse transcription reaction was carried out using SuperScript™ III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer’s instructions. Real-time PCR analysis was performed on the 7900HT Fast Real-Time PCR system (Applied Biosystems) at standard reaction conditions using Power SYBR Green PCR Master Mix (Applied Biosystems). Briefly, after a 2 min denaturation at 95 °C, 40 cycles carried out at 95 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s. Relative mRNA levels calculated with the ∆∆CT method were analyzed using SDS Version 2.4.1 software. Data presented are from an experiment with three replicates. Results and Discussion A set of known and as yet not understood molecular mechanisms regulate the expression of genes at (post-) transcription and (post-) translation stages in eukaryotic cells. It is generally agreed that there are three main routes of influence including; 1) combinatorial fashion of regulators and DNA 2) modulation through interaction of a control factor with the transcription machinery and 3) epigenetic modifications, are responsible for regulating gene transcription in various cell types. As a result of these mechanisms, the Sox2 gene is differentially expressed in a variety of embryonic cells and adult differentiated cell types. We choose the Sox2 gene for testing TALE-TF functionality in cattle fibroblast cells due to its well-known promoter structure as well as the lack of the evidence of methylation in the immediate regulatory regions. Our data shows that the Sox2 gene already has a basal level of expression in cattle fibroblast cells, indicating that Sox2 promoter has some activity in bovine fibroblast cells. As a proof of concept, we first tested activity of Sox2-TALE-TF in HEK293 cells. Florescent microscopy results showed that minCMV promoter has high level of activity in cattle fibroblast so it would not be considered as a suitable reporter of TALE-TF functionality. Analysis of qRT-PCR results for sox2 gene in cattle fibroblast, however, showed that Sox2 expression has been increased by 3.529 fold changes in transfected cells. Several mammalian genes have reportedly been targeted with TALE-TFs, however, fold changes in abundance of targeted gene transcripts rarely is above 5 times when individual TALE-TFs were tested. A number of factors affecting TALE-TFs activity have been reported, however, due to cell type specific chromatin state, multiple cis- and trans-acting regulatory elements and epigenetic modifications are involved in the regulation of gene expression, therefore it is very difficult to predict the efficacy of a TALE-TFs without experimental validation. Conclusion In summary current study indicated that TALE-TFs as artificial transcription factors have the ability to increase the endogenous expression of genes in cattle fibroblast cells.