Avoidance of reporter assay distortions from fused dual reporters.

Avoidance of reporter assay distortions from fused dual reporters.
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避免融合双报告基因导致报告基因分析失真。

DOI:
10.1261/rna.061051.117
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发表时间:
2017-08
期刊:
RNA (New York, N.Y.)
影响因子:
--
通讯作者:
Atkins JF
Atkins JF
中科院分区:
其他
文献类型:
--
作者:
Loughran G;Howard MT;Firth AE;Atkins JF

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在融合的报告基因之间定位测试序列允许在测试序列之前和之后监测翻译水平和成帧。许多研究,包括那些重新编码,如生产性核糖体移码和终止密码子通读,使用可区分的内切酶或荧光蛋白作为报告。由于测试序列产物干扰单个报告分子活性或稳定性而导致的偶尔扭曲,在这里证明可以通过引入测试序列侧翼的串联StopGo序列(2A)来避免。使用这种新的载体系统(pSGDluc),我们为使用3′茎环刺激器进行ACP 2通读提供了证据,但未能检测到报道的VEGFA通读。
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 ACP2 readthrough, but failed to detect the reported VEGFA readthrough.
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发表时间: 2010-01-01
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影响因子: --
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