| Improving CRISPR Gene Editing Efficiency by Proximal dCas9 Targeting
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Author:
Date:
2017-08-05
[Abstract] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems function as an adaptive immune system in bacteria and archaea for defense against invading viruses and plasmids (Barrangou and Marraffini, 2014). The effector nucleases from some class 2 CRISPR-Cas systems have been repurposed for heterologous targeting in eukaryotic cells (Jinek et al., 2012; Cong et al., 2013; Mali et al., 2013; Zetsche et al., 2015). However, the genomic environments of eukaryotes are distinctively different from that of prokaryotes in which CRISPR-Cas systems have evolved. Mammalian heterochromatin was found to be a barrier to target DNA access by Streptococcus pyogenes Cas9 (SpCas9), and nucleosomes, the basic units of ...
[摘要] 集群定期间隔短回归重复（CRISPR）和CRISPR相关（Cas）系统作为细菌和古菌中的适应性免疫系统，用于防御入侵病毒和质粒（Barrangou和Marraffini，2014）。来自某些2类CRISPR-Cas系统的效应核酸酶已被重新用于真核细胞中的异源靶向（Jinek et al。，2012; Cong等人，2013; Mali ，2013; Zetsche等人，2015）。然而，真核生物的基因组环境与CRISPR-Cas系统发展的原核生物的基因组环境有明显的不同。发现哺乳动物异染色质是通过化脓性链球菌Cas9（SpCas9）靶向DNA接近的障碍，并且还发现染色质的基本单位的核小体阻碍了通过SpCas9的靶DNA进入和切割[ （Knight等人，，2015; Hinz等人，2015; Horlbeck等人，2016年） ; ...
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| 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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| Preparation of Recombinant Galectin-3 for Cancer Studies
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Author:
Date:
2016-01-05
[Abstract] Galectin-3 is a member of a class of proteins termed Galectins, characterized by their ability to bind glycans containing β-galactose (Cummings and Liu, 2009). Galectin-3 binds preferentially to proteoglycans terminating with N-acetyllactosamine (LacNAc) chains (i.e., tandem repeats of galactose) (Newlaczyl and Yu, 2011). Galectin-3 is unique among the galectins in its chimeric structure. It shares a conserved carbohydrate recognition domain (CRD) with the other galectins, but has a long amino-terminal tail that is thought to be involved in protein aggregation. It can also form homodimers through its CRD (Cummings and Liu, 2009). Galectin-3 has been found to have diverse functions in tumorigenesis including: signaling, apoptosis inhibition, immune suppression, cell growth, and ...
[摘要] Galectin-3是称为Galectins的一类蛋白质的成员，其特征在于它们结合含有β-半乳糖的聚糖的能力（Cummings和Liu，2009）。 Galectin-3优先结合以N-乙酰乳糖胺（LacNAc）链（即半乳糖的串联重复）终止的蛋白聚糖（Newlaczyl和Yu，2011）。 Galectin-3在其嵌合结构中的半乳凝素中是独一无二的。它与其他半乳凝素共享保守的碳水化合物识别结构域（CRD），但具有被认为参与蛋白质聚集的长氨基末端尾部。它也可以通过CRD形成同型二聚体（Cummings和Liu，2009）。已经发现Galectin-3在肿瘤发生中具有多种功能，包括：信号转导，凋亡抑制，免疫抑制，细胞生长和转移等。 ...
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