Cell Type-specific Gene Expression Profiling in the Mouse Liver

Summary

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Abstract

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah^-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Introduction

As the main metabolic organ in vertebrates, the liver is responsible for glucose homeostasis, serum protein synthesis, bile acid secretion, and xenobiotic metabolism and detoxification. The liver possesses an extraordinary capacity to regenerate the injured parenchyma upon exposure to toxins to prevent immediate liver failure¹. However, failure of regeneration can occur in the setting of acetaminophen or alcohol overconsumption, which can lead to acute liver failure². Furthermore, chronic liver injury caused by viral hepatitis infection, fatty liver disease, and steatohepatitis can cause liver fibrosis, cirrhosis, and he....

Protocol

All methods that involve the use of mice are consistent with the guidelines provided by the IACUC of the Penn Office of Animal Welfare at the University of Pennsylvania.

1. Reagent Preparation

1. Cycloheximide
   1. To make 500 µL of 0.1 g/mL cycloheximide, suspend 50 mg of cycloheximide in 500 µL of methanol.
      CAUTION: Cycloheximide is extremely toxic to the environment and can cause congenital malformation. All wastes and buffers containing cycloheximide should be collected for proper disposal.
      NOTE: Cycloheximide can be stored at 4 °C for up t....

Results

To profile gene expression in repopulating hepatocytes of the Fah^-/- mouse, Gfp:Rpl10a fusion and Fah transgenes are co-delivered within a transposon-containing plasmid⁸ (TRAP vector) to livers by hydrodynamic injection (Figure 1A). The removal of nitisinone induces a toxic liver injury that creates a selection pressure for hepatocytes stably expressing FAH to repopulate the injure.......

Discussion

TRAP-seq is a technique for cell type-specific isolation of translating mRNA via epitope-tagged ribosomes and presents an alternative to FACS approaches, as it circumvents limitations such as time requirements of FACS⁹. Instead, TRAP allows rapid and efficient isolation of RNA directly from bulk tissues, helping to avoid any alterations in gene expression. TRAP-seq is especially well-suited for use in the repopulating Fah^-/- mouse liver, as hepatocyte expansion following remova.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 mL Tissue Grinder, Potter-Elv, Coated	DWK Life Sciences (Wheaton)	358007
Absolutely RNA Miniprep Kit	Agilent	400800
Anti-GFP antibodies	Memorial Sloan-Kettering Antibody & Bioresource Core	GFP Ab #19C8 and GFP Ab #19F7
Bovine Serum Albumin, IgG-Free, Protease-Free	Jackson ImmunoResearch	001-000-162
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail	Roche	11836170001
Cycloheximide	Millipore Sigma	C7698
Deoxycholic acid, DOC	Millipore Sigma	D2510
D-Glucose, Dextrose	Fisher Scientific	D16
DL-Dithiothreitol	Millipore Sigma	D9779
Dynabeads MyOne Streptavidin T1	Thermo Fisher Scientific	65602
Fisherbrand Petri Dishes with Clear Lid	Fisher Scientific	FB0875712
HBSS (10x), calcium, magnesium, no phenol red	Thermo Fisher Scientific	14065-056
HEPES, 1 M Solution, pH 7.3, Molecular Biology Grade, Ultrapure, Thermo Scientific	Thermo Fisher Scientific	AAJ16924AE
Magnesium chloride, MgCl2	Millipore Sigma	M8266
Methanol	Fisher Scientific	A452
NanoDrop 2000/2000c Spectrophotometer	Thermo Fisher Scientific	VV-83061-00
NEBNext Poly(A) mRNA Magnetic Isolation Module	New England BioLabs	E7490S
NEBNext Ultra RNA Library Prep Kit for Illumina	New England BioLabs	E7530S
Nonylphenyl polyethylene glycol, NP-40. IGEPAL CA-630	Millipore Sigma	I8896
Nuclease-Free Water, not DEPC-Treated	Ambion	AM9932
Overhead Stirrer	DWK Life Sciences (Wheaton)	903475
PBS Buffer (10x), pH 7.4	Ambion	AM9625
Pierce Recombinant Protein L, Biotinylated	Thermo Fisher Scientific	29997
Potassium chloride, KCl	Millipore Sigma	P4504
RNA 6000 Pico Kit & Reagents	Agilent	5067-1513
RNaseZap RNase Decontamination Solution	Invitrogen	AM9780
RNasin Ribonuclease Inhibitors	Promega	N2515
Sodium azide, NaN3	Millipore Sigma	S2002
Sodium bicarbonate, NaHCO3	Millipore Sigma	S6297
SUPERase·In RNase Inhibitor	Invitrogen	AM2694