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.
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Acknowledgements
This protocol is the in vivo evolution of the in vitro RNAi screen set up by the Agami, Beijersbergen and Bernards laboratories, which are acknowledged. We are thankful to the NKI Genomics Core (R. Kerkhoven and I. de Rink), as well as to Microscopy, Screening (B. Morris), FACS and Animal Facilities for assistance with experiments, and to the following persons for expert advice: M. Amendola (virus packaging and titration), P. Possik and K. Greig (amplification and barcode-tagging of shRNA libraries) and J. Jaspers (tumor cell isolation). We are grateful to K. Sutherland and A. Berns (both from the Netherlands Cancer Institute) for KrasG12D/+;Trp53−/− cells and B. Siteur for excellent assistance with animal experiments, as well as to W. Akhtar, C. Biancotto, A. Gruszka and E. Guccione for critical input on this manuscript. This work was supported by the European Commission project (Marie Curie) FCMOG (FP7-PEOPLE-2009-IEF 251938) and Fondazione Lorini to G.G., by the Koningin Wilhelmina Fonds (KWF) project NKI2013-6030 to G.G. and M.v.L., and by the Netherlands Genomics Initiative to M.v.L.
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Contributions
G.G. designed and developed the procedure and wrote the manuscript. M.S., D.H. and M.C. helped with experiments and data analysis. M.v.L. supervised the study.
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Integrated supplementary information
Supplementary Figure 1 Example of NSCLC cell grafting efficiency.
Mouse lung adenocarcinoma cells with a KrasG12D;Trp53-/- were modified with a library containing both (candidate) TSG and oncogenes and luciferase emission was acquired immediately after tail vein injection (∼30 min), 24 hours and 7 days later. a) Luciferase emission for each mouse back and belly at day 0, 1 and 7. Statistics used one-way ANOVA and Dunn test for multiple comparisons. b) The IVIS scan for two independent animals from (a) is shown at the indicated days.
All animal experiments were conducted in compliance with the European Union guidelines for the use and care of laboratory animals and were reviewed and approved by the animal ethics committee (DEC) of the Netherlands Cancer Institute.
Supplementary Figure 2 Input tables.
Example of table formatting for Box 4 and Step 49.
Supplementary Figure 3 Example of hairpin Sanger sequencing result as validation for MOI<1 infection.
Brain tumour generated with a pool of shRNA was dissociated and plated at clonal density to isolate clonal cell populations (glioma-spheres). Multiple single spheres (>20) were subjected to hairpin library amplification followed by Sanger sequencing. Successful base calling during shRNA sequencing indicates the presence of one single hairpin per cell is shown as an example (as in 6). Reproduced from ref. 6, © 2013, with permission from Elsevier.
Supplementary Figure 4 Example of correlation between two in vivo dropout screen biological replicas.
Each data point represents one normalized hairpin enrichment/depletion value in each experimental group (recipient animals #1-#2-#3-#4-#5 are correlated with #6-#7-#8-#9-#10). Data were processed as in step 48, linear regression was performed as in step 55, and axes variation is -15 to 10, lower left quadrant include dropout hairpins in KrasG12V/+;Trp53-/- cells. All animal experiments were conducted in compliance with the European Union guidelines for the use and care of laboratory animals and were reviewed and approved by the animal ethics committee (DEC) of the Netherlands Cancer Institute
Supplementary Figure 5 Example of experimental variation between hairpins.
For two different “Input” cell types and libraries, we have sequenced using a depth of ∼5,000 fold the library size and the actual reads count (Log2 transformed) is shown. These results reflect the actual variation introduced by experimental steps 1–30.
Supplementary information
Supplementary Figures 1–5
Supplementary Figures 1–5 (PDF 671 kb)
Supplementary Software: Perl script file example.
Contains the set of instructions to perform annotation of the sequencing. It should be copied into a text file and named “harpin_fast.pl” or a different name should be indicated in the perl script. Moreover, we indicated the name “barcode.txt” in case of multiplexed runs: this file is not required for the perl script to function, but it should be renamed in perl script if a different name is used for that instruction. (TXT 2 kb)
Supplementary Table 1: Example of input annotation table.
This file contains the relevant information to run Supplementary Data 1, and can be used as a template for customized hairpin libraries. It should be copied into a text file and named “Perl harpin_fast.pl annotation_file.txt”. (TXT 101 kb)
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Gargiulo, G., Serresi, M., Cesaroni, M. et al. In vivo shRNA screens in solid tumors. Nat Protoc 9, 2880–2902 (2014). https://doi.org/10.1038/nprot.2014.185
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DOI: https://doi.org/10.1038/nprot.2014.185
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