避免融合双报告基因导致的报告基因检测失真。
Avoidance of reporter assay distortions from fused dual reporters.
作者信息
Loughran Gary, Howard Michael T, Firth Andrew E, Atkins John F
机构信息
School of Biochemistry and Cell Biology, University College Cork, Cork T12 YT57, Ireland.
Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA.
出版信息
RNA. 2017 Aug;23(8):1285-1289. doi: 10.1261/rna.061051.117. Epub 2017 Apr 25.
Abstract
Positioning test sequences between fused reporters permits monitoring of both translation levels and framing, before and after the test sequence. Many studies, including those on recoding such as productive ribosomal frameshifting and stop codon readthrough, use distinguishable luciferases or fluorescent proteins as reporters. Occasional distortions, due to test sequence product interference with the individual reporter activities or stabilities, are here shown to be avoidable by the introduction of tandem StopGo sequences (2A) flanking the test sequence. Using this new vector system (pSGDluc), we provide evidence for the use of a 3' stem-loop stimulator for readthrough, but failed to detect the reported readthrough.
摘要
将测试序列置于融合报告基因之间，可以监测测试序列前后的翻译水平和读框。许多研究，包括那些关于重新编码的研究，如生产性核糖体移码和终止密码子通读，都使用可区分的荧光素酶或荧光蛋白作为报告基因。由于测试序列产物干扰单个报告基因的活性或稳定性而导致的偶尔失真，在这里表明通过在测试序列两侧引入串联的StopGo序列（2A）是可以避免的。使用这种新的载体系统（pSGDluc），我们提供了使用3'茎环刺激物进行通读的证据，但未能检测到报道的通读情况。
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1
Evidence for Pro-angiogenic Functions of VEGF-Ax.
Cell. 2016 Sep 22;167(1):275-284.e6. doi: 10.1016/j.cell.2016.08.054.
2
Ribosomal readthrough at a short UGA stop codon context triggers dual localization of metabolic enzymes in Fungi and animals.
PLoS Genet. 2014 Oct 23;10(10):e1004685. doi: 10.1371/journal.pgen.1004685. eCollection 2014 Oct.
3
Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals.
Elife. 2014 Sep 23;3:e03640. doi: 10.7554/eLife.03640.
4
Evidence of efficient stop codon readthrough in four mammalian genes.
Nucleic Acids Res. 2014 Aug;42(14):8928-38. doi: 10.1093/nar/gku608. Epub 2014 Jul 10.
5
Programmed translational readthrough generates antiangiogenic VEGF-Ax.
Cell. 2014 Jun 19;157(7):1605-18. doi: 10.1016/j.cell.2014.04.033.
6
Methods for studying IRES-mediated translation of positive-strand RNA viruses.
Methods. 2013 Feb;59(2):167-79. doi: 10.1016/j.ymeth.2012.09.004. Epub 2012 Sep 23.
7
Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation.
Plant Cell Rep. 2012 Oct;31(10):1877-87. doi: 10.1007/s00299-012-1301-5. Epub 2012 Jul 10.
8
A novel approach for directing transgene expression in Drosophila: T2A-Gal4 in-frame fusion.
Genetics. 2012 Mar;190(3):1139-44. doi: 10.1534/genetics.111.136291. Epub 2011 Dec 29.
9
2A peptides provide distinct solutions to driving stop-carry on translational recoding.
Nucleic Acids Res. 2012 Apr;40(7):3143-51. doi: 10.1093/nar/gkr1176. Epub 2011 Dec 2.
10
Evidence of abundant stop codon readthrough in Drosophila and other metazoa.
Genome Res. 2011 Dec;21(12):2096-113. doi: 10.1101/gr.119974.110. Epub 2011 Oct 12.
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