Name: cell type-specific gene expression profiling in the mouse liver
Uploaded: 2019-09-17T14:30:00
Description: Watch this Scientific Journal Video about Cell Type-specific Gene Expression Profiling in the Mouse Liver at JoVE.com

This work was supported by the following grants: F31-DK113666 (AWW), K01-DK102868 (AMZ), K08-DK106478 (KJW), and P30-DK050306 (Pilot grant to KJW).

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
<ol>
	<li>Cycloheximide
	<ol>
		<li>To make 500 &#181;L of 0.1 g/mL cycloheximide, suspend 50 mg of cycloheximide in 500 &#181;L of methanol. 
		CAUTION: Cycloheximide is extremely toxic to the environment and can cause congenital malformation. All wastes and buff...

<ol>
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C., Taner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+transplantation:+Current+status+and+challenges.">Liver transplantation: Current status and challenges.</a> World Journal of Gastroenterology. 22 (18), 4438-4445 (2016).</li><li>Michalopoulos, G. K., DeFrances, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> Science. 276 (5309), 60-66 (1997).</li><li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration+after+partial+hepatectomy:+critical+analysis+of+mechanistic+dilemmas.">Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas.</a> The American Journal of Pathology. 176 (1), 2-13 (2010).</li><li>Grompe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+fumarylacetoacetate+hydrolase+is+responsible+for+the+neonatal+hepatic+dysfunction+phenotype+of+lethal+albino+mice.">Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice.</a> Genes &amp; Development. 7 (12A), 2298-2307 (1993).</li><li>Wangensteen, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+facile+method+for+somatic,+lifelong+manipulation+of+multiple+genes+in+the+mouse+liver.">A facile method for somatic, lifelong manipulation of multiple genes in the mouse liver.</a> Hepatology. 47 (5), 1714-1724 (2008).</li><li>Wang, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRAP-seq+identifies+cystine/glutamate+antiporter+as+a+driver+of+recovery+from+liver+injury.">TRAP-seq identifies cystine/glutamate antiporter as a driver of recovery from liver injury.</a> The Journal of Clinical Investigation. 128 (6), 2297-2309 (2018).</li><li>Heiman, M., Kulicke, R., Fenster, R. J., Greengard, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+mRNA+purification+by+translating+ribosome+affinity+purification+(TRAP).">Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP).</a> Nature Protocols. 9 (6), 1282-1291 (2014).</li><li>Agilent Technologies. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Agilent RNA 6000 Pico: User Manual. , (2016).</li><li>NEBNext. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NEBNext Ultra RNA Library Prep Kit for Illumina: User Manual. , (2018).</li><li>NanoDrop. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2000/2000c Spectrophotomerter: User Manual. , (2009).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+translational+profiling+in+acute+kidney+injury.">Cell-specific translational profiling in acute kidney injury.</a> The Journal of Clinical Investigation. 124 (3), 1242-1254 (2014).</li><li>Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glutathione+synthesis.">Regulation of glutathione synthesis.</a> Molecular Aspects of Medicine. 30 (1-2), 42-59 (2009).</li><li>Wang, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamic+chromatin+architecture+of+the+regenerating+liver.">The dynamic chromatin architecture of the regenerating liver.</a> bioRxiv. , (2019).</li><li>Zahm, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-content+in+vivo+screen+to+identify+microRNA+epistasis+in+the+repopulating+mouse+liver.">A high-content in vivo screen to identify microRNA epistasis in the repopulating mouse liver.</a> bioRxiv. , (2019).</li><li>Stork, C., Zheng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Profiling+of+RNA-Protein+Interactions+Using+CLIP-Seq.">Genome-Wide Profiling of RNA-Protein Interactions Using CLIP-Seq.</a> Methods in Molecular Biology. 1421, 137-151 (2016).</li><li>Zhao, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+antioxidant+pathway+activity+and+reserve+determine+toxicity+and+specificity+of+the+biliary+toxin+biliatresone+in+zebrafish.">Glutathione antioxidant pathway activity and reserve determine toxicity and specificity of the biliary toxin biliatresone in zebrafish.</a> Hepatology. 64 (3), 894-907 (2016).</li><li>Halpern, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+spatial+reconstruction+reveals+global+division+of+labour+in+the+mammalian+liver.">Single-cell spatial reconstruction reveals global division of labour in the mammalian liver.</a> Nature. 542 (7641), 352-356 (2017).</li><li>Camp, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+communication+regulates+human+liver+bud+development+from+pluripotency.">Multilineage communication regulates human liver bud development from pluripotency.</a> Nature. 546 (7659), 533-538 (2017).</li><li>Wang, Y. J., Kaestner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+RNA-Seq+of+the+Pancreatic+Islets--a+Promise+Not+yet+Fulfilled?.">Single-Cell RNA-Seq of the Pancreatic Islets--a Promise Not yet Fulfilled?.</a> Cell Metabolism. 29 (3), 539-544 (2019).</li></ol>

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...

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 hepatocellular carcinoma3. The only available curative treatment for end-stage liver disease is transplantation but is limited by organ shortage, preventing efficient treatment for all patients4. A better understanding of the recovery process after toxic liver injury is therefore crucial for the development of treatments to stimulate regeneration sufficient to rescue function in the diseased organ.
The most broadly applied model system for the study of liver regeneration is partial hepatectomy in rodents, in which a large proportion of the liver is resected to stimulate rapid hepatocyte expansion5. However, partial hepatectomy does not recapitulate hepatocyte expansion following toxic liver injury due to the lack of immune cell infiltration and hepatocyte cell necrosis often observed in the setting of acute liver injury in humans6. A more suitable system to model this form of organ renewal is the Fah-/- mouse, which lacks functional fumarylacetoacetate hydrolase (FAH) required for proper tyrosine metabolism and develops severe liver damage leading to death7. These mice can be maintained in a healthy state indefinitely by treatment with the drug nitisinone in the drinking water. Alternatively, FAH expression can be restored by transgene delivery to a subset of hepatocytes, which will expand to repopulate the liver upon nitisinone removal8.
To profile the gene expression changes of repopulating hepatocytes, a tool to specifically isolate replicating hepatocytes in the Fah-/- mouse without contamination from the neighboring injured hepatocytes and other cell types is required. Unfortunately, fluorescence-assisted cell sorting (FACS) of hepatocytes is difficult since (1) the fragility of repopulating hepatocytes leads to poor recovery after liver perfusion, (2) replicating hepatocytes are highly variable in size, making isolation of a pure population by FACS difficult, and (3) the procedure time from liver perfusion to RNA isolation is greater than 2 h, hence gene expression profiles may undergo substantial artificial changes before samples are acquired9.
Alternatively, the expression of epitope-tagged ribosomes specifically in repopulating hepatocytes allows for the rapid isolation of actively translating mRNA bound by ribosomes using affinity purification immediately after organ harvest, with bulk liver tissue lysates. Here, we describe a protocol to perform translating-ribosome affinity purification (TRAP)10 followed by high-throughput RNA-sequencing (TRAP-seq), to specifically isolate and profile mRNA in repopulating hepatocytes in the Fah-/- mouse9. Coexpression of green fluorescent protein-tagged ribosomal protein (GFP:RPL10A) with FAH allows affinity purification of translating mRNA bound by polysomes containing GFP:RPL10A. This method avoids any cell dissociation steps, such as liver perfusion to isolate fragile repopulating hepatocytes. Instead, it utilizes lysis of whole organ tissue and antibodies to rapidly extract the RNA specifically from the target cells. Finally, isolation of abundant, high-quality mRNA via TRAP-seq enables downstream applications such as sequencing analysis to profile the dynamic change of gene expression during the repopulation process.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Tissue Grinder, Potter-Elv, Coated</td>
			<td>DWK Life Sciences (Wheaton)</td>
			<td>358007</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolutely RNA Miniprep Kit</td>
			<td>Agilent</td>
			<td>400800</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFP antibodies</td>
			<td>Memorial Sloan-Kettering Antibody &#38; Bioresource Core</td>
			<td colspan="2">GFP Ab #19C8 and GFP Ab #19F7</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, IgG-Free, Protease-Free</td>
			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Millipore Sigma</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxycholic acid, DOC</td>
			<td>Millipore Sigma</td>
			<td>D2510</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose, Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Millipore Sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin T1</td>
			<td>Thermo Fisher Scientific</td>
			<td>65602</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Petri Dishes with Clear Lid</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (10x), calcium, magnesium, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>14065-056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, 1M Solution, pH 7.3, Molecular Biology Grade, Ultrapure, Thermo Scientific</td>
			<td>Thermo Fisher Scientific</td>
			<td>AAJ16924AE</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride, MgCl2&#160;</td>
			<td>Millipore Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>VV-83061-00</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Poly(A) mRNA Magnetic Isolation Module</td>
			<td>New England BioLabs</td>
			<td>E7490S</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Ultra RNA Library Prep Kit for Illumina</td>
			<td>New England BioLabs</td>
			<td>E7530S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonylphenyl polyethylene glycol, NP-40. IGEPAL CA-630</td>
			<td>Millipore Sigma</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-Free Water, not DEPC-Treated</td>
			<td>Ambion</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>Overhead Stirrer</td>
			<td>DWK Life Sciences (Wheaton)</td>
			<td>903475</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Buffer (10x), pH 7.4</td>
			<td>Ambion</td>
			<td>AM9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Recombinant Protein L, Biotinylated</td>
			<td>Thermo Fisher Scientific</td>
			<td>29997</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride, KCl</td>
			<td>Millipore Sigma</td>
			<td>P4504</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA 6000 Pico Kit &#38; Reagents</td>
			<td>Agilent</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap RNase Decontamination Solution</td>
			<td>Invitrogen</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin Ribonuclease Inhibitors</td>
			<td>Promega</td>
			<td>N2515</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide, NaN3</td>
			<td>Millipore Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, NaHCO3</td>
			<td>Millipore Sigma</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERase&#183;In RNase Inhibitor</td>
			<td>Invitrogen</td>
			<td>AM2694</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell type-specific gene expression profiling in the mouse liver

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.

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...

Watch this Scientific Journal Video about Cell Type-specific Gene Expression Profiling in the Mouse Liver at JoVE.com

Cell Type-specific Gene Expression Profiling in the Mouse Liver

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.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

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