کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6491244 | 43404 | 2015 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Highly sensitive luciferase reporter assay using a potent destabilization sequence of calpain 3
ترجمه فارسی عنوان
با استفاده از یک توالی بی ثبات کننده قوی از کالپین 3، گزارشگر حساس لوسیفراز بسیار حساس است
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کلمات کلیدی
NF-κBcharged-coupled deviceCalpain 3CAPN3eGFPCCDFBSDMEMTNFαDulbecco's modified Eagle's medium - Medal of Eagle اصلاح شده Dulbeccotumor necrosis factor α - تومور نکروز عامل αthymidine kinase - تیمیدین کینازfetal bovine serum - سرم جنین گاوnuclear factor-κB - فاکتور هسته ای κBfluorescence in situ hybridization - فلورسانس در هیبریداسیون در محلluciferase - لوسیفرازFish - ماهیReal-time monitoring - نظارت بر زمان واقعیenhanced green fluorescent protein - پروتئین فلورسنت سبز افزایش یافته است
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
چکیده انگلیسی
Reporter assays that use luciferases are widely employed for monitoring cellular events associated with gene expression in vitro and in vivo. To improve the response of the luciferase reporter to acute changes of gene expression, a destabilization sequence is frequently used to reduce the stability of luciferase protein in the cells, which results in an increase of sensitivity of the luciferase reporter assay. In this study, we identified a potent destabilization sequence (referred to as the C9 fragment) consisting of 42 amino acid residues from human calpain 3 (CAPN3). Whereas the half-life of Emerald Luc (ELuc) from the Brazilian click beetle Pyrearinus termitilluminans was reduced by fusing PEST (t1/2 = 9.8 to 2.8 h), the half-life of C9-fused ELuc was significantly shorter (t1/2 = 1.0 h) than that of PEST-fused ELuc when measurements were conducted at 37 °C. In addition, firefly luciferase (luc2) was also markedly destabilized by the C9 fragment compared with the humanized PEST sequence. These results indicate that the C9 fragment from CAPN3 is a much more potent destabilization sequence than the PEST sequence. Furthermore, real-time bioluminescence recording of the activation kinetics of nuclear factor-κB after transient treatment with tumor necrosis factor α revealed that the response of C9-fused ELuc is significantly greater than that of PEST-fused ELuc, demonstrating that the use of the C9 fragment realizes a luciferase reporter assay that has faster response speed compared with that provided by the PEST sequence.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biotechnology - Volume 194, 20 January 2015, Pages 115-123
Journal: Journal of Biotechnology - Volume 194, 20 January 2015, Pages 115-123
نویسندگان
Mayu Yasunaga, Kazutoshi Murotomi, Hiroko Abe, Tomomi Yamazaki, Shigeaki Nishii, Tetsuya Ohbayashi, Mitsuo Oshimura, Takako Noguchi, Kazuki Niwa, Yoshihiro Ohmiya, Yoshihiro Nakajima,