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Updated: Jul 31, 2026
MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells
Published on: December 21, 2011
In vivo shRNA screens in solid tumors
Gaetano Gargiulo1, Michela Serresi1, Matteo Cesaroni2
1Division of Molecular Genetics, the Netherlands Cancer Institute, Amsterdam, the Netherlands.
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.
Insights
This study presents a protocol for RNA interference (RNAi) screens in mouse models to identify genes involved in tumor development. The method uses pooled shRNA libraries for efficient screening of multiple genes in solid tumors.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Loss-of-function (LOF) experiments are crucial for understanding gene roles in tumorigenesis.
- Pooled shRNA libraries enable high-throughput screening of multiple genes simultaneously.
- RNAi screens in animal models utilize shRNAs for gene disruption and cell fate barcoding.
Purpose of the Study:
- To provide a detailed protocol for performing RNAi screens in orthotopic mouse tumor models.
- To offer guidelines for applying RNAi to a wide range of solid tumors.
- To highlight key considerations for designing and executing effective RNAi studies.
Main Methods:
- Assembly and lentiviral packaging of shRNA libraries.
- Transduction of tumor-initiating cells (TICs) with shRNA constructs.
- In vivo transplantation, tumor DNA recovery, sequencing, and data analysis.
Main Results:
- Identification of tumor suppressors and oncogenes.
- Dissection of biological pathways involved in tumorigenesis.
- Feasible application in glioma and lung adenocarcinoma models within 6-9 weeks.
Conclusions:
- The protocol facilitates efficient LOF screening of multiple genes in solid tumors.
- This approach aids in identifying novel therapeutic targets and understanding cancer biology.
- The method is adaptable for diverse tumor types and research questions.
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