کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
10826606 1064804 2005 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Stabilization and analysis of intron lariats in vivo
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Stabilization and analysis of intron lariats in vivo
چکیده انگلیسی
The analysis of lariats produced in vivo during pre-mRNA splicing is a powerful tool for elucidation of regulatory mechanisms and identification of natural recursive splicing events. Nevertheless, this analysis is technically challenging because lariats normally have short half-lives. With appropriate controls, RT-PCR amplification and sequencing of the region spanning the 2′-5′ phosophodiester bond at the branch junction can be a sensitive and versatile method for lariat analysis. This approach can be facilitated and enhanced by reducing the activity of debranching enzyme (DBR) in order to stabilize lariats. We have generated a set of plasmids for dsRNA-mediated knockdown of DBR under diverse conditions in transgenic Drosophila and in cultured cells. We describe the use of these plasmids and protocols for lariat analysis. We have generated transgenic Drosophila strains carrying a GAL4-regulated RNAi construct that allows selective knockdown of DBR in specific tissues or developmental stages, using the large collection of available GAL4 expression lines. These strains should prove useful for detailed developmental analyses of alternative and recursive splicing and for genetic analyses of splicing factors. Similar approaches should be readily adaptable to other organisms.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Methods - Volume 37, Issue 4, December 2005, Pages 368-375
نویسندگان
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