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公司名称: Thermo Fisher Scientific
产品编号: 30391023
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RNA ImmunoGenic Assay: A Method to Detect Immunogenicity of in vitro Transcribed mRNA in Human Whole Blood
Author:
Date:
2020-12-20
[Abstract]  The mRNA therapeutics is a new class of medicine to treat many various diseases. However, in vitro transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important aspects of the human immune system is still required. Here, we present a simple ex vivo method called ‘RNA ImmunoGenic Assay’ to measure immunogenicity of IVT-mRNAs in human whole blood. Chemically modified and unmodified mRNA ... [摘要]  [摘要] mRNA疗法是治疗多种疾病的新型药物。然而,我Ñ体外转录(IVT)的mRNA触发由于人类内体和细胞质RNA传感器,但是修饰的核苷的掺入识别的免疫应答已经示出吨ö减少此类反应。牛逼herefore ,测定标志着重要的环节人体免疫系统仍然需要。这里,我们提出一个简单的离体称为“RNA方法免疫ģ ENIC测定”测量的IVT-mRNA的免疫原性小号在人全血。将化学修饰和未修饰的mRNA与转染试剂(TransIT )复合,并在人全血中共同孵育。特异性细胞因子测定(TNF- α ,INF- α ,INF- γ ,IL-6和IL-12p70的),使用的ELISA。进行qPCR分析以揭示特异性免疫途径的激活。所述RNA免疫ģ ENIC测定提供小号的简单且快速的方法来检测供体特异性-针对mRNA的治疗剂的免疫应答。

图形摘要:

RNA免疫基因测定的示意图

[背景] mRNA治疗是基因治疗的重要一类(Sahin等,2014 ;Antony等,2015 ...

Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line
Author:
Date:
2017-05-20
[Abstract]  There are several in silico programs that endeavor to predict the functional impact of an individual’s sequence variation at splice donor/acceptor sites, but experimental confirmation is problematic without a source of RNA from the individual that carries the variant. With the aid of an exon trapping vector, such as pSPL3, an investigator can test whether a splice site sequence change leads to altered RNA splicing, through expression of reference and variant mini-genes in mammalian cells and analysis of the resultant RNA products. [摘要]  有几个计算机程序尝试预测个体在剪接供体/受体位点的序列变异的功能影响,但实验确认是有问题的,没有携带变体的个体的RNA来源。借助于外显子捕获载体,例如pSPL3,研究人员可以通过在哺乳动物细胞中表达参考和变体小基因来测试剪接位点序列变化是否导致改变的RNA剪接,并分析所得RNA产物。

背景 我们希望通过实验测试在TEK基因中鉴定的两个剪接供体位点变体c.760 + 2T> C和c.3300 + 2delT的功能影响(Souma等人,2016)。通常情况下,携带这些序列变体的个体不能获得细胞或mRNA的样品,因此我们利用外显子捕获方法作为功能测试。来自患者的DNA样品可用于感兴趣的基因组区域的PCR扩增。如果患者gDNA样品不可用,也可以通过诸如基于PCR的定点诱变等方法将序列变体并入野生型序列。
 外显子捕获方法最初是为了鉴定长期基因组DNA中的未知外显子而开发的(Duyk等人,1990)。创建了pSPL3外显子捕获载体以提高外显子鉴定的效率和可靠性,并且还允许筛选更大的基因组片段(Church et al。,1994; Nisson等,1994)。 pSPL3载体含有由SV40启动子组成的小型人造基因,具有功能性剪接供体和受体位点的外显子 - 内含子 - ...

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