In vivo shRNA screens in solid tumors

Nat Protoc. 2014 Dec;9(12):2880-902. doi: 10.1038/nprot.2014.185. Epub 2014 Nov 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / genetics
  • Adenocarcinoma of Lung
  • Animals
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Female
  • Gene Library
  • Gene Targeting / methods*
  • Genetic Techniques*
  • Heterografts
  • Humans
  • Lung Neoplasms / genetics
  • Male
  • Mice, Inbred BALB C
  • Neoplasms, Experimental / genetics*
  • Oncogenes
  • RNA Interference
  • RNA, Small Interfering / genetics*
  • Transfection

Substances

  • RNA, Small Interfering