In vivo gain-of-function cDNA library screening for colonization genes in a mouse model of pulmonary metastasis.
In vivo gain-of-function cDNA library screening for colonization genes in a mouse model of pulmonary metastasis.
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DOI：
10.1016/j.xpro.2022.101413
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发表时间：
2022-06-17
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我们提供了一种功能获得（GOF） cDNA筛选基因的方案，这些基因促进癌细胞在继发组织中定植，这是转移级联的最后也是最致命的一步。我们提出了cDNA病毒文库制备和递送技术，从而在基于du145的肺转移小鼠模型中恢复促进定植的序列。适用于其他cDNA文库和癌症模型，这种方法将被证明在列举转移性定植的内在遗传决定因素方面广泛有用。有关使用和执行本协议的完整详情，请参阅。定植是转移级联的最后一个也是最致命的瓶颈步骤，功能获得（GOF） cDNA筛选适用于检测肺DU145细胞的癌症定植，这些细胞含有GOF前列腺cDNA文库，产生肺部大转移，大菌落的基因组PCR暴露插入的潜在定植基因的cDNA。我们提供了一种功能获得（GOF） cDNA筛选基因的方案，这些基因促进癌细胞在继发组织中定植，这是转移级联的最后也是最致命的一步。我们提出了cDNA病毒文库制备和递送技术，从而在基于du145的肺转移小鼠模型中恢复促进定植的序列。适用于其他cDNA文库和癌症模型，这种方法将被证明在列举转移性定植的内在遗传决定因素方面广泛有用。
We provide a protocol for gain-of-function (GOF) cDNA screen of genes that foster cancer cell colonization of secondary tissues, the last and most lethal step of the metastasis cascade. We present techniques for cDNA viral library preparation and delivery leading up to the recovery of colonization-promoting sequences in a proof-of-concept DU145-based mouse model of pulmonary metastasis. Adapted to other cDNA libraries and cancer models, this approach would prove widely useful in enumerating intrinsic genetic determinants underlying metastatic colonization. For complete details on the use and execution of this protocol, please refer to. Colonization is the last and most lethal bottleneck step of the metastasis cascade Gain-of-function (GOF) cDNA screen was adapted to probe cancer colonization of lung DU145 cells harboring GOF prostate cDNA library generated pulmonary macro-metastases Genomic PCR of macro-colonies exposed inserted cDNAs of potential colonization genes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. We provide a protocol for gain-of-function (GOF) cDNA screen of genes that foster cancer cell colonization of secondary tissues, the last and most lethal step of the metastasis cascade. We present techniques for cDNA viral library preparation and delivery leading up to the recovery of colonization-promoting sequences in a proof-of-concept DU145-based mouse model of pulmonary metastasis. Adapted to other cDNA libraries and cancer models, this approach would prove widely useful in enumerating intrinsic genetic determinants underlying metastatic colonization.