کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10532745 | 961709 | 2012 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Full-length 3â²-untranslated region reporter construction with recombineering
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
Complexity in higher animals derives in part from various modalities of protein-coding gene expression regulation, including microRNA repression by binding to 3â²-untranslated regions (UTRs) of specific genes. Reporter constructs containing candidate microRNA target sites are a popular approach of functional studies, and full-length 3â²-UTR sequences are preferred because they contain all regulatory elements and preserve higher order structure as much as possible. However, this approach is often handicapped by the extreme length of the 3â²-UTR. Here, we present a rapid and accurate cloning procedure to generate full-length 3â²-UTR reporter constructs by recombinogenic engineering (recombineering) in vivo cloning. The approach includes making retrieval constructs by sequence- and ligation-independent cloning (SLIC) and retrieving the full-length 3â²-UTR in one exon to the retrieval construct from a bacterial artificial chromosome (BAC) by recombineering to generate the final full-length 3â²-UTR reporter construct for the gene of interest. This method is successfully implemented with mouse full-length 3â²-UTRs of Igf1 (6.5Â kb), Igf1r (7.5Â kb), and Sp1 (5.5Â kb). Expansion of this method is adaptable to retrieve 3â²-UTRs encoded in more than one exon by removing the introns from the BAC first with recombineering. This method will advance functional studies of regulation of gene expression at the post-transcriptional level through microRNA suppression.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 424, Issue 2, 15 May 2012, Pages 162-167
Journal: Analytical Biochemistry - Volume 424, Issue 2, 15 May 2012, Pages 162-167
نویسندگان
Ruqiang Liang, Eugenia Wang,