Generation of Helper Plasmids Encoding Mutant Adeno-associated Virus Type ۲ Capsid Proteins with Increased Resistance against Proteasomal Degradation
- سال انتشار: 1392
- محل انتشار: مجله علوم پایه پزشکی ایران، دوره: 16، شماره: 7
- کد COI اختصاصی: JR_IJBMS-16-7_001
- زبان مقاله: انگلیسی
- تعداد مشاهده: 293
نویسندگان
Shahroud University of Medical Sciences, Shahroud, Iran
Stem Cell and Regenerative Medicine Research Department, Iranian Academic Center for Education, Culture and Research (ACECR), Mashhad Branch, Mashhad, Iran
Department of Cardiology, Leiden University Medical Center, Leiden, the Netherlands
Department of Cardiology, Leiden University Medical Center, Leiden, the Netherlands
Department of Cardiology, Leiden University Medical Center, Leiden, the Netherlands
چکیده
Objective(s): Adeno-associated virus type ۲ (AAV۲) vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV۲ vector helper plasmids encoding capsid proteins with single, double or triple Y→F mutations. Materials and Methods: A one-step, high-fidelity polymerase chain reaction (PCR) cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV۲ cap genes encoding VP۱ mutants with Y→F substitutions in residues ۴۴۴, ۵۰۰ or ۷۳۰. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y۴۴۴۵۰۰F mutant), subcloning (Y۴۴۴۷۳۰F and Y۵۰۰۷۳۰F mutants) or a combination of both techniques (Y۴۴۴۵۰۰۷۳۰F mutant). Results: Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV۲ cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV۲ vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping. Conclusion: PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV۲ vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.کلیدواژه ها
Gene therapy Viral vector Adeno-associated virus type ۲ Capsid protein Proteasomal degradation Mutagenesis Transduction efficiencyاطلاعات بیشتر در مورد COI
COI مخفف عبارت CIVILICA Object Identifier به معنی شناسه سیویلیکا برای اسناد است. COI کدی است که مطابق محل انتشار، به مقالات کنفرانسها و ژورنالهای داخل کشور به هنگام نمایه سازی بر روی پایگاه استنادی سیویلیکا اختصاص می یابد.
کد COI به مفهوم کد ملی اسناد نمایه شده در سیویلیکا است و کدی یکتا و ثابت است و به همین دلیل همواره قابلیت استناد و پیگیری دارد.