CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells

被引：18

作者：

Baillat, David ^{[1
]}

Russell, William K. ^{[1
]}

Wagner, Eric J. ^{[1
]}

机构：

[1] Univ Texas Med Branch, Dept Biochem & Mol Biol, Galveston, TX 77550 USA

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2016年 / 128卷

关键词：

CRISPR-mediated purification; Integrator complex; Mass spectrometry; RNA-POLYMERASE-II; GENOME; NUCLEASES; SPECIFICITY; ACTIVATION; EXPRESSION; EFFICIENCY; BIOLOGY; CAS9;

D O I：

10.1016/j.pep.2016.08.011

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination. Finally, using silver staining, Western blotting and LC-MS/MS we compare the components of INT of purifications from CRISPR derived lines to 293T cells overexpressing FLAG-INTS11 to define a highly resolved constituency of mammalian INT. (C) 2016 Elsevier Inc. All rights reserved.

引用

页码：101 / 108

页数：8

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