Figure 1. TRAP enables cell type–specific isolation of RNA from quiescent and repopulating hepatocytes.

(A) The approach for isolating repopulating hepatocyte RNA with the Fah^–/– model involves use of the FAH expression construct to mediate liver repopulation and the GFP-tagged ribosomal protein L10a (GFP-L10a) to specifically isolate translating mRNAs with TRAP. Injection of the Rosa^LSL-GFP-L10a mouse with the AAV8-TBG-Cre virus, which has a tropism for hepatocytes and has a hepatocyte-specific promoter driving Cre expression in nearly all hepatocytes, allows for immunoprecipitation of translating mRNA from quiescent hepatocytes. (B) Bioanalyzer tracings of affinity-purified RNA from mice treated with or without the TRAP vector. FU, fluorescence units. (C) Representative (n = 3) IHC images of GFP show progressive repopulation over time in Fah^–/– mice as well as complete labeling of quiescent hepatocytes in Rosa^LSL-GFP-L10a mice 1 week after injection of AAV8-TBG-Cre. No GFP expression was observed in livers from the uninjected mice. IF of Ki67 and GFP confirmed successful liver repopulation in Fah^–/– mice injected with the TRAP vector, as all Ki67-positive hepatocytes express GFP. IF costaining also showed global GFP-expressing and rare Ki67-positive hepatocytes, indicating that the control tissue was truly quiescent. Note that a subset of mice showed only partial repopulation at 4 weeks (4-week regeneration after severe injury). Scale bars: 1 mm (top) and 100 μm (bottom).