title: In vivo shRNA screens in solid tumors.
abstract: Loss-of-function (LOF) experiments targeting multiple genes during tumorigenesis can be implemented using pooled shRNA libraries. RNAi screens in animal models rely on the use of multiple shRNAs to simultaneously disrupt gene function, as well as to serve as barcodes for cell fate outcomes during tumorigenesis. Here we provide a protocol for performing RNAi screens in orthotopic mouse tumor models, referring to glioma and lung adenocarcinoma as specific examples. The protocol aims to provide guidelines for applying RNAi to a diverse spectrum of solid tumors and to highlight crucial considerations when designing and performing these studies. It covers shRNA library assembly and packaging into lentiviral particles, and transduction into tumor-initiating cells (TICs), followed by in vivo transplantation, tumor DNA recovery, sequencing and analysis. Depending on the target genes and tumor model, tumor suppressors and oncogenes can be identified or biological pathways can be dissected in 6-9 weeks.
authors.affiliation: Division of Molecular Genetics, the Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Gaetano
authors.identifier: 0000000154144251
authors.initials: G
authors.lastname: Gargiulo
authors.affiliation: Division of Molecular Genetics, the Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Michela
authors.initials: M
authors.lastname: Serresi
authors.affiliation: Fels Institute, Temple University School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Matteo
authors.initials: M
authors.lastname: Cesaroni
authors.affiliation: Division of Molecular Genetics, the Netherlands Cancer Institute, Amsterdam, the Netherlands.
authors.forename: Danielle
authors.initials: D
authors.lastname: Hulsman
authors.affiliation: 1] Division of Molecular Genetics, the Netherlands Cancer Institute, Amsterdam, the Netherlands. [2] Cancer Genomics Centre, the Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: van Lohuizen
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics; D000077192:Adenocarcinoma of Lung; D000818:Animals; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005260:Female; D015723:Gene Library; D018390:Gene Targeting / Q000379:methods*; D005821:Genetic Techniques; D064593:Heterografts; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D008297:Male; D008807:Mice, Inbred BALB C; D009374:Neoplasms, Experimental / Q000235:genetics*; D009857:Oncogenes; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
chemical_list: D034741:RNA, Small Interfering