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Gallenkamp IOI400.XX2.C refrigerated shaking incubator

公司名称: Gallenkamp
产品编号: IOI400.XX2.C
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Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi
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2017-05-20
[Abstract]  To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3’ end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3’ end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of ... [摘要]  为了实现克氏锥虫内源蛋白的C末端标记,我们使用Cas9 / pTREX-n载体(Lander等,2015)在3'末端插入特异性标签序列(3xHA或3xc-Myc)特定的感兴趣基因(GOI)。将靶向GOI 3'末端的嵌合sgRNA进行PCR扩增,并克隆到Cas9 / pTREX-n载体中。然后扩增通过同源重组诱导DNA修复的DNA供体分子。该供体序列包含标签序列和抗生素抗性标记,加上对应于位于终止密码子右上游的区域和在GOI基因座的Cas9靶位点下游的100bp同源臂。载体pMOTag23M(Oberholzer等,2006)或pMOHX1Tag4H(Lander等,2016b)用作DNA供体扩增的PCR模板。用sgRNA / Cas9 / pTREX-n构建体和DNA供体盒共转染的Epimastigotes然后用抗生素培养5周以选择双重抗性寄生虫。最终通过PCR和Western印迹分析验证了内源基因标记。
【背景】自从CRISPR / ...

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