A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells
| العنوان: | A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells |
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| المؤلفون: | Rinji Akada, Mikiko Nakamura, Hisashi Hoshida, Tohru Yarimizu, Ryo Iwakiri, Junko Akada, Ayako Suzuki |
| المصدر: | Molecular Biotechnology. 57:767-780 |
| بيانات النشر: | Springer Science and Business Media LLC, 2015. |
| سنة النشر: | 2015 |
| مصطلحات موضوعية: | Gene Expression, Bioengineering, Biology, Transfection, Polymerase Chain Reaction, Applied Microbiology and Biotechnology, Biochemistry, chemistry.chemical_compound, Gene expression, Escherichia coli, Animals, Humans, Enhancer, Molecular Biology, Gene, DNA Primers, Terminator Regions, Genetic, Oligonucleotide, Molecular biology, Recombinant Proteins, HEK293 Cells, Terminator (genetics), chemistry, Rabbits, Primer (molecular biology), DNA, HeLa Cells, Plasmids, Biotechnology |
| الوصف: | Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures. |
| تدمد: | 1559-0305 1073-6085 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0ab202d861a26df3499967fe18f26d10 https://doi.org/10.1007/s12033-015-9870-5 |
| حقوق: | CLOSED |
| رقم الأكسشن: | edsair.doi.dedup.....0ab202d861a26df3499967fe18f26d10 |
| قاعدة البيانات: | OpenAIRE |
Find this article in full text from Springer Verlag. | تدمد: | 15590305 10736085 |
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