دورية أكاديمية
Studies of fluorescent imaging for mRNA detection in living cells
| العنوان: | Studies of fluorescent imaging for mRNA detection in living cells |
|---|---|
| المؤلفون: | Wang, Zhenghui |
| المصدر: | All Theses and Dissertations (ETDs) |
| بيانات النشر: | Washington University Open Scholarship |
| سنة النشر: | 2011 |
| المجموعة: | Washington University St. Louis: Open Scholarship |
| مصطلحات موضوعية: | Chemistry, Biochemistry, Fluorescence, Imaging, mRNA, PNA |
| الوصف: | This dissertation focuses on the study of imaging mRNA in living cells. To achieve this research objective, three approaches have been utilized:: 1) Imaging of a transgenic mRNA tagged by multiple repeats of malachite green: MG) binding aptamer.: 2) Imaging of inducible nitric oxide synthase: iNOS) mRNA by strand-displacement activated Peptide Nucleic Acid: PNA) probes.: 3) Imaging of iNOS mRNA by binary fluorescently labeled PNA probes. The first approach was based on the work of our former lab member Dr. Huafeng Fang, who had constructed a multiple MG binding aptamer tagged transgene: Flag-mβ2AR-GFP-MGVI), which could also express a green fluorescence protein associated with an adrenergic receptor protein. It has been reported that the tagged aptamer sequence can increase the fluorescence of MG up to 2000 fold by binding to MG. Total RNA extract of the transfected MDCK cells has shown up to 22 times increase of fluorescence in the presence of MG. Confocal fluorescence imaging study has shown that in the presence of MG, cells expressing the transgene showed both the fluorescence of GFP and enhanced fluorescence of MG. A flow cytometry study detected that in the presence of MG and transfected cells showed 1.3 fold increase of fluorescence compared to the wild type MDCK cells. The next approach was to use strand-displacement activated PNA probes to detect the iNOS mRNA in living RAW 264.7 mouse macrophage cells. A probe constitutes of an antisense 23-mer fluorescein: FAM) labeled antisense PNA and a 17-mer Dabcylplus labeled complementary DNA was used. The fluorescence of the FAM was quenched when the two strands hybridized to each other. In the presence of target mRNA, the shorter strand was displaced by the mRNA, which has more base pairs complementary to the PNA. The fluorescence of FAM was restored and thus could be used to detect the mRNA. The probe has been shown to be able to detect the target DNA and in vitro transcribed mRNA in solution. Fluorescence in situ hybridization: FISH) showed that the probes ... |
| نوع الوثيقة: | text |
| وصف الملف: | application/pdf |
| اللغة: | English unknown |
| العلاقة: | https://openscholarship.wustl.edu/etd/663Test; https://openscholarship.wustl.edu/cgi/viewcontent.cgi?article=1662&context=etdTest |
| الإتاحة: | https://openscholarship.wustl.edu/etd/663Test https://openscholarship.wustl.edu/cgi/viewcontent.cgi?article=1662&context=etdTest |
| رقم الانضمام: | edsbas.4943E96F |
| قاعدة البيانات: | BASE |
| الوصف غير متاح. |