| Assaying the Effects of Splice Site Variants by Exon Trapping in a Mammalian Cell Line
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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。
＆NBSP;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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| Transwell Co-culture of Bone Marrow Macrophages with Tumor Cells
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Author:
Date:
2014-05-05
[Abstract] Bone is a primary site of metastasis from prostate and breast cancers. Bone marrow macrophages (BMMs) are mediators of inflammatory processes and are thought to promote tumor growth in the skeletal sites. In order to elucidate how their interactions with tumor cells impact aggressiveness of metastatic tumors in bone in vitro methods are required. By employing a system in which BMMs and tumor cells are grown separately, yet share the media and exchange soluble factors, contribution of each cell type in the context of BMM-tumor cell relationship in the bone marrow can be investigated. ...
[摘要] 骨是来自前列腺和乳腺癌的原发转移部位。骨髓巨噬细胞（BMM）是炎症过程的介质，并且被认为促进骨骼部位中的肿瘤生长。为了阐明它们与肿瘤细胞的相互作用如何影响骨髓中转移性肿瘤的侵袭性，需要体外方法。通过采用其中BMM和肿瘤细胞分开生长，但共享培养基和交换可溶性因子的系统，可以研究在骨髓中BMM-肿瘤细胞关系的上下文中每种细胞类型的贡献。该系统的另外的优点包括研究以下的能力：1）共培养时BMM和肿瘤细胞中的表型改变; 2）蛋白质和基因表达的细胞特异性调节;和3）对增殖和细胞存活的影响。值得注意的是，该transwell共培养系统不限于BMM和肿瘤细胞，并且可以容易地修饰以包括骨髓微环境的其他组分（例如，脂肪细胞，基质细胞，成骨细胞）。
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| Packaging of Retroviral RNA into Viral Particles Analyzed by Quantitative Reverse Transcriptase-PCR
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Author:
Date:
2013-04-20
[Abstract] Formation of viral particles and packaging of genomic retroviral RNA into these particles are important steps in the late phase of the viral replication cycle. The efficiency of the incorporation of viral or cellular RNAs into viral particles can be studied using a quantitative Reverse Transcriptase-PCR (RT-qPCR)-based approach. After isolation of cytoplasmic RNA from either infected or transfected cells and extraction of virus particle-associated RNA, specific RNA levels present in both fractions are determined. The ratio of virion-associated and cytoplasmic RNA defines the encapsidation ...
[摘要] 病毒颗粒的形成和基因组逆转录病毒RNA包装到这些颗粒中是病毒复制周期晚期的重要步骤。 可以使用定量逆转录酶-PCR（RT-qPCR）为基础的方法研究将病毒或细胞RNA掺入病毒颗粒中的效率。 在从感染的或转染的细胞分离细胞质RNA并提取病毒颗粒相关的RNA后，测定两种级分中存在的特异性RNA水平。 病毒体相关的和细胞质RNA的比率定义了衣壳化效率（Brandt等人，2007; Blissenbach等人，2010; Grewe等人，/em>，2012）。
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