Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

Marta H. Kubala; Yves A. DeClerck

doi:10.3791/56333

JoVE Journal
Cancer Research

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JoVE Journal Cancer Research

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

肿瘤细胞系基因表达的条件性击倒研究单核/巨噬细胞在微环境中的招募

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November 23, 2017

DOI: 10.3791/56333-v

Marta H. Kubala^1,2, Yves A. DeClerck^1,2,3

¹Division of Hematology, Oncology and Blood and Marrow Transplantation, Department of Pediatrics,University of Southern California, ²The Saban Research Institute of Children's Hospital Los Angeles, ³Department of Biochemistry and Molecular Medicine,University of Southern California

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本议定书作为一个计划, 建立一个功能性的癌症细胞系的系统, 并随后使用, 特别是研究肿瘤细胞源性蛋白质在招募单核/巨噬细胞到肿瘤微环境的作用。

该方案的总体目的是利用癌细胞中基因表达的条件性敲低来研究单核细胞和巨噬细胞在体外向肿瘤微环境的募集。这种方法可以帮助回答肿瘤微环境领域中关于癌细胞和免疫系统细胞之间交叉存量性质的关键问题。该技术的主要优点是此处使用的系统需要单载体克隆，允许快速生成多个克隆，并且表现出非常低的厚度。

还展示了一种无需直接接触抗体即可纯化人单核细胞的方法，该方法避免了意外激活，同时产生超过 95% 纯度的人单核细胞群。为了产生病毒颗粒，用 25 μg pLKO-tet-on shRNA、25 μg psPAX 包装质粒和 5 μg 包膜表达质粒 pMD2 转染 70% 汇合的 150 mm 培养皿 HEK-293 细胞。G 按照标准方案使用转染试剂。

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