In vivo shRNA screens 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.