دانشگاه اراک سلول و بافت 2228-7035 16 3 2025 09 23 Creation of a lentiviral recombinant construct containing the PDX1 gene using the CRISPR/Cas9 system ایجاد یک سازه لنتی ویروسی نوترکیب حاوی ژن PDX1 کلون شده با استفاده از سیستم CRISPR/Cas9 277 290 731366 10.61882/JCT.16.3.277 FA عظیمه اخلاق پور استادیار، گروه سلول‫های بنیادی و سرطان، دانشکده زیست فناوری، دانشگاه تخصصی فناوری‫های نوین آمل، آمل، ایران.‬‬‬‬‬‬‬‬‬‬‬‬ محسن بصیری دکتری تخصصی، گروه سلول‫های بنیادی و زیست شناسی تکوینی، مرکز تحقیقات علوم سلولی، موسسه زیست شناسی و فناوری سلول‫های بنیادی رویان، تهران، ایران‬‬‬‬‬‬‬‬‬‬‬‬ مریم مبارک قمصری استادیار، گروه زیست فناوری میکروبی، دانشکده زیست فناوری، دانشگاه تخصصی فناوری‫های نوین آمل، آمل، ایران‬‬‬‬‬‬ سیده نفیسه حسنی دانشیار، گروه سلول‫های بنیادی و زیست شناسی تکوینی، مرکز تحقیقات علوم سلولی، موسسه زیست شناسی و فناوری سلول‫های بنیادی رویان، تهران، ایران‬‬‬‬‬‬‬‬‬‬‬‬ Journal Article Introduction: Homeodomain transcription factor 1 (PDX-1) is necessary for proper pancreatic development and β-cell function. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, in conjunction with CRISPR-associated protein 9 (Cas9), facilitates modifications, particularly deletions, within the genome. The use of lentiviral vectors for gene transfer to somatic stem cells has increased significantly. They harbor advantageous features, including a relatively high gene loading capacity, a diminished likelihood of gene expression silencing following integration into the genome, and high efficiency in delivering genes to a variety of target cells. Furthermore, they elicit no undesirable immune reactions and offer a broad selection of alternative envelope proteins. Aim: This study aims to introduce a mutation in the chicken PDX-1 gene. A guide RNA (gRNA) will be designed to complement a specific region of the PDX-1 gene. This approach aims to create targeted mutations with potential therapeutic applications in gene therapy. Materials and Methods: The PDX-1 gene was cloned using the LentiCRISPRv2GFP vector. To delete and modify the PDX-1 gene, a specific single guide RNA (sgRNA) sequence is designed. This sgRNA sequence is approximately 100 base pairs (bp) in length, with about 76 bp derived from the scaffold segment already integrated into the LentiCRISPRv2GFP vector. The region responsible for binding the sgRNA to the target genomic sequence is 20 nucleotides long and is complementary to 20 nucleotides in exon one of the PDX-1 gene. The CRISPR Design Tool (http://crispr.mit.edu) was used to design a specific sgRNA sequence targeting the PDX1 locus (sgPDX1). The double-stranded oligonucleotide fragment and the LentiCRISPRv2GFP vector were subjected to enzymatic digestion using the BsmbI restriction enzyme. To create the LentiCRISPRv2GFP-sgPDX1 vector, the cleaved sgPDX1 sequence was joined with the cleaved vector. This was accomplished by mixing the vector and nucleotide fragment and subjecting them to a ligation reaction using the T4 ligase enzyme. The resulting vector contains a transcript of the sgRNA, specifically sgPDX1. The heat shock method was employed to transform Escherichia coli Top10 host cells with the cloned LentiCRISPRv2GFP-sgPDX1 vector. To confirm the presence of the sgPDX-1 gene in the vector, a polymerase chain reaction (PCR) was conducted using primers targeted at a portion of the U6 promoter sequence (forward primer) and a segment downstream of the sgRNA gene region (reverse primer). Additionally, each PCR product was analyzed using enzymatic digestion with EcoRV and BsmBI enzymes. To confirm the successful cloning, the PCR product was sequenced. Results: The PCR results showed an amplified fragment measuring 267 base pairs (bp), which indicated the presence of the sgPDX-1 gene. This was visualized on an agarose gel. Additionally, the reverse digestion with two restriction enzymes produced a 2000 bp fragment isolated from the vector. The sequencing results confirmed that the cloning process was successful. Discussion: Genome editing technology has been highlighted in bird species due to their significant potential for economic production and creating models for biological research. The application of CRISPR gene editing technology in avian research has yielded promising advancements, particularly in the modification of somatic cells and tissues in birds. By precisely targeting primordial germ cells (PGCs), researchers can implement specific genetic modifications within the chick genome, thereby facilitating the development of genetically edited chicks. Numerous studies have investigated the role of the PDX-1 gene in gene therapy using the CRISPR/Cas9 system across various organisms. However, this study is the first to design and produce a recombinant construct containing the chicken PDX1 gene using a lentiviral vector with the CRISPR/Cas9 construct. This advancement aims to create a practical research platform for future studies by enabling the direct transfer of this recombinant construct into chick embryos and cells. Conclusion: The chicken PDX-1 gene was cloned into a lentiviral vector containing the CRISPR/Cas9 construct. The recombinant vector LentiCRISPRv2GFP-sgPDX1 was successfully obtained. The gene construct developed in this study has significant potential for investigating gene function and for use in gene therapy. Consequently, utilizing this construct may enable the creation of an animal model in chicken embryos featuring defects, including an empty organ or tissue cavity. Such a model will allow for the investigation and treatment of the defects using specific drugs and cell lines. <span dir="RTL" lang="FA" style="font-size: 11.0pt; mso-ansi-font-size: 10.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-hansi-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">هدف: <span dir="RTL" lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">هدف از این مطالعه، ایجاد موتاسیون در ژن <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">PDX<span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">-1<span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;"> جوجه، از طریق ایجاد یک <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">gRNA<span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;"> مکمل با قسمتی از ژن <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">PDX-1<span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;"> است. هدف این رویکرد ایجاد جهش‌های هدفمند با کاربردهای درمانی بالقوه در ژن‌درمانی است.<span lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;"> <span dir="RTL" lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">مواد و روش‌ها: <span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">جهت حذف و تغییرات در ژن <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">PDX-1<span dir="RTL" style="font-size: 10.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;"> <span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">جوجه، توالی <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">RNA<span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;"> راهنمای تکی <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">(single guide RNA, sgRNA<span style="font-size: 11.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">)<span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;"> از نرم افزار اینترنتی <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">CRISPR Design Tool<span dir="RTL" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;"> طراحی شد. و از طریق آنزیم‌های برشی و واکنش الحاق در وکتور لنتی ویروسی <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">LentiCRISPRv2GFP<span dir="RTL" lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; color: black; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;"> کلون شد و از طریق شوک حرارتی در سلول‌های میزبان <span style="font-size: 11.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">Escherichia coli Top10<span dir="RTL" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;"> <span dir="RTL" lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">ترانسفورم <span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">ش<span dir="RTL" lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">د. <span lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;"> <span dir="RTL" lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">نتایج<span dir="RTL" lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">: <span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">نتایج <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">PCR<span dir="RTL" style="font-size: 10.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;"> <span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">یک قطعه تکثیر شده به<span style="font-size: 11.0pt; line-height: 115%; font-family: 'Calibri',sans-serif; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-hansi-theme-font: minor-latin; mso-bidi-font-family: Arial; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">‫<span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">طول ۲۶۷ جفت باز <span dir="RTL" lang="FA" style="font-size: 10.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">(<span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">bp<span dir="RTL" lang="FA" style="font-size: 10.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">) <span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">را نشان داد که نشان‌دهنده وجود ژن <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;">sgPDX-1<span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA; mso-no-proof: yes;"> بود. این قطعه روی ژل آگارز قابل مشاهده بود. علاوه بر این، هضم معکوس با دو آنزیم محدودکننده، یک قطعه ۲۰۰۰ جفت بازی جدا شده از ناقل را تولید کرد. نتایج تعیین توالی، موفقیت‌آمیز بودن فرآیند کلونینگ را تایید کرد. <span dir="RTL" lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">نتیجه<span style="font-size: 11.0pt; line-height: 115%; font-family: 'Calibri',sans-serif; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-hansi-theme-font: minor-latin; mso-bidi-font-family: Arial; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">‫<span dir="RTL" lang="AR-SA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">گیری: <span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">ژن <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">PDX-1<span dir="RTL" style="font-size: 10.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;"> <span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">مرغ در یک وکتور لنتی‌ویروسی حاوی ساختار <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">CRISPR/Cas9<span dir="RTL" style="font-size: 10.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;"> <span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">کلون شد. وکتور نوترکیب <span style="font-size: 10.0pt; line-height: 115%; font-family: 'Times New Roman',serif; mso-fareast-font-family: Calibri; mso-bidi-font-family: 'B Nazanin'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">LentiCRISPRv2GFP-sgPDX1<span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;"> با موفقیت به<span style="font-size: 11.0pt; line-height: 115%; font-family: 'Calibri',sans-serif; mso-ascii-theme-font: minor-latin; mso-fareast-font-family: Calibri; mso-fareast-theme-font: minor-latin; mso-hansi-theme-font: minor-latin; mso-bidi-font-family: Arial; mso-bidi-theme-font: minor-bidi; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">‫<span dir="RTL" lang="FA" style="font-size: 11.0pt; line-height: 115%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-font-kerning: 1.0pt; mso-ligatures: standardcontextual; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">دست آمد. ساختار ژنی توسعه‌یافته در این مطالعه پتانسیل قابل توجهی برای بررسی عملکرد ژن و استفاده در ژن‌درمانی دارد. در نتیجه، استفاده از این ساختار ممکن است امکان ایجاد یک مدل حیوانی در جنین‌های مرغ با نقص، از جمله حفره خالی اندام یا بافت، را فراهم کند. چنین مدلی امکان بررسی و درمان نقص‌ها را با استفاده از داروها و رده‌های سلولی خاص فراهم می‌کند.

https://jct.araku.ac.ir/article_731366_a690278a4dfda825ae7ceb6286647dd7.pdf