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Review
. 2015 May;36(5):315-22.
doi: 10.1016/j.it.2015.03.007. Epub 2015 Apr 27.

In vivo RNAi screens: concepts and applications

Shane Crotty  1 , Matthew E Pipkin  2
Affiliations
Review

In vivo RNAi screens: concepts and applications

Shane Crotty et al. Trends Immunol. 2015 May.

Abstract

Functional genomics approaches that leverage the RNAi pathway have been applied in vivo to examine the roles of hundreds or thousands of genes; mainly in the context of cancer. Here, we discuss principles guiding the design of RNAi screens, parameters that determine success and recent developments that have improved accuracy and expanded the applicability of these approaches to other in vivo settings, including the immune system. We review recent studies that have applied in vivo RNAi screens in T cells to examine genes that regulate T cell differentiation during viral infection, and that control their accumulation in tumors in a model of adoptive T cell therapy. In this context, we put forward an argument as to why RNAi approaches in vivo are likely to provide particularly salient insight into immunology.

Keywords: T follicular helper cell differentiation; immunotherapy; in vivo RNAi screens; memory CD8 T cell differentiation; pooled screens.

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Figures

Figure 1
Figure 1. In vivo screen approaches using pooled shRNA DNAs delivered to cells
(A) Screens based on pooling shRNA DNAs. DNA encoding tens to tens-of-thousands of viral (or transposon) delivered shRNAs is pooled. (B) Pooled shRNA DNA is transfected into viral packaging cells to generate viral supernatants, and used to transduce query cells (tumors, or tissue progenitors), or is transferred in utero to transduce developing fetal tissue progenitor cells [51], at a low multiplicity of infection (MOI) to promote integration of one shRNA clone per cell. Alternatively, pooled DNA is injected (transposon-mediated) into recipient mice to directly deliver shRNAs to target tissues (e.g., liver) [36]. (C) Sequencing methods are used to quantify representation of proviral DNA encoding shRNA sequences in libraries prepared from DNA extracted from the input pool at time zero, as compared to libraries prepared from DNA extracted from tumors, regenerated tissue, or normal tissue to determine differential selection of cells bearing shRNAs that induce functional effects.
Figure 2
Figure 2. A pooled approach to in vivo T cell screens using arrayed transduction of shRNAs
(A) Screens based on pooling cells after transduction with unique shRNAs in arrays. DNA for viral shRNA constructs is used to package virus and transduce query cells at high-efficiency (high MOIs) in arrayed format and cells are pooled before transfer in vivo [10]. (B) FACS-based recovery of all transferred cells after subsetting based on differentiation-associated phenotypes. (C) Next generation sequencing libraries amplified from proviral DNA encoding shRNA sequences are prepared from genomic DNA samples of input and sorted cell populations and differential shRNA representation is quantified after high-throughput sequencing.
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