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Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In vivo

Biochemistry

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Summary

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

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Moran, P., Guo, Y., Yuan, R., Barnekow, N., Palmer, J., Beck, A., Ren, B. Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In vivo. J. Vis. Exp. (147), e59624, doi:10.3791/59624 (2019).

Abstract

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues. 

Introduction

In complex tissues such as the mammalian brain, heart and lung, the high levels of cellular heterogeneity complicate the analysis of gene expression data derived from whole tissue samples. To observe gene expression profiles in a particular cell type in vivo, a new methodology has been developed recently, which allows the interrogation of the entire translated mRNA complement of any genetically defined cell type. This methodology is known as the translating ribosome affinity purification (TRAP) technique1,2. It is a useful tool to study endothelial cell biology and angiogenesis when combined with genetically manipulating other angiogenesis-associated genes in animals. 

We have shown that angiogenic PKD-1 signaling and the transcription of angiogenic gene CD36 are critical for endothelial cell (EC) differentiation and functional angiogenesis3,4,5,6. To determine molecular mechanisms of angiogenic and metabolic signaling in gene transcription and EC transdifferentiation, we have created genetically engineered TRAP mice with specifically deleted angiogenic genes on the basis of TRAP technique1,2. Furthermore, in our TRAP animals, not only do they have pkd-1 or cd36 gene deficiency in the vascular endothelia or global deletion of cd36 gene, but an enhanced green fluorescence protein (EGFP) is also genetically tagged onto EC’s translating ribosomes. TRAP permits affinity purification of ribosome-bound mRNA directly from the vascular endothelia of targeted tissues, enabling the analysis of gene expression and identification of new transcriptomes that are associated with EC differentiation and angiogenesis directly under in vivo conditions. We have successfully isolated ribosome-bound RNA from the endothelia in these genetically engineered animals. The purified RNA can be used for further characterization of angiogenic or arteriogenic genes in the regulation of EC differentiation and functions. This protocol provides a step-by-step guide to implement the TRAP approach for the isolation of mRNA in ECs directly in vivo.

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Protocol

For animal experiments, all methods described here have been approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

1. Prepare reagents

  1. Prepare lysis buffer to concentrations of 10 mM HEPES, pH 7.4, 150 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 100 mg/mL cycloheximide, protease inhibitors, and recombinant RNase inhibitors to concentrations as described below.
    1. Add following reagents to 500 mL of RNase-free deionized water: 1.19 g of HEPES, 5.59 g of KCl, 0.24 g of MgCl2, 35 mg of DTT, 0.5 mL of cycloheximide, and NaOH as needed until pH 7.4, EDTA-free protease inhibitors (one mini tablet per 10 mL) and RNase inhibitor (10 µL/mL).
    2. Store in a 4 °C fridge for up to 1 month.
  2. Prepare a high-salt polysome wash buffer to concentrations of 10 mM HEPES, pH 7.4, 350 mM KCl, 5 mM MgCl2, 1% vol/vol CA-630, 0.5 mM DTT, and 100 mg/mL cycloheximide.
    1. Add following reagents to 500 mL of RNase-free deionized water: 1.19 g of HEPES, 13.05 g of KCl, 0.24 g of MgCl2, 5 mL of nonionic, non-denaturing detergent, 5 of 7.7 mg tubes of DTT, and 0.5 mL of cycloheximide, and NaOH as needed until pH 7.4.
    2. Store in 4 °C fridge for up to 1 month.
  3. Bind anti-GFP antibody to Protein G magnetic beads prior to starting experiment.
    1. Add 10 mg of anti-GFP antibody diluted in 200 mL of PBS to Protein G beads.
    2. Incubate with end over end rotation for 10 minutes at room temperature.
    3. Place the beads on a magnetic rack and remove the supernatant.
    4. Suspend the beads in 200 mL of PBS and store in 4 °C fridge for up to 1 week.
  4. Prepare ice-cold PBS with 100 mg/mL cycloheximide.
    1. Add 1 volume of cycloheximide solution (100 mg/mL) to 99 volumes of ice-cold PBS.

2. Isolate and lyse desired tissues

  1. Euthanize mice by IP injection of ketamine (500 mg/kg/body weight) and xylazine (10 mg/kg/body weight) and isolate desired tissues (i.e., heart, lung). Immediately proceed to next step.
  2. Place desired tissues into 500 mL of ice-cold PBS with 100 mg/mL cycloheximide.
  3. Mince tissue into a cell suspension with a motor-driven homogenizer or a small-clearance glass homogenizer. If using a motor-driven homogenizer, limit homogenization to less than 1 minute at low frequency (<15,000 Hz) to avoid RNA denaturation.
  4. Suspend cell pellet in 200 mL of lysis buffer by pipetting and redrawing up buffer several times. Further homogenize cell suspension with 10 strokes in a small-clearance glass homogenizer or for 15 seconds at low frequency (<15,000 Hz) in a motor driven homogenizer.
  5. Centrifuge homogenates for 10 min at 2,000 x g at 4 °C to pellet nuclei and large cell debris, and keep the supernatant.
  6. Add nonionic, non-denaturing detergent to 1% vol/vol and DHPC to 30 mM to the supernatant. Incubate on ice for 5 min.
  7. Centrifuge lysate for 10 min at 16,000 x g to pellet insoluble material. Transfer and keep 15% of clear lysate as input for future steps.

3. Isolate ribosome/mRNA complexes

  1. Add 50 mL of antibody-bound beads to cell-lysate supernatant and incubate mixture at 4 °C with end-over-end rotation for 30 min. This is where the anti-GFP antibodies will bind the GFP-tagged ribosomes, allowing us to further isolate the RNA from these ribosomes.
  2. Collect beads on a magnetic rack and wash 5 times with high-salt polysome wash buffer.
    1. Draw up and discard liquid once beads have collected on the side of the tube. Then pipette and redraw up 200 mL of high-salt polysome wash buffer several times. Repeat this step 5 times and discard all buffer following final repetition. Immediately proceed to next step.

4. Isolate mRNA

  1. Place beads in RLT buffer. The following steps are taken directly from the RNeasy mini kit protocol and were not expanded on in any way.
    CAUTION: RLT buffer contains guanidine salts; do NOT mix with bleach.
  2. Centrifuge lysate for 3 min at full speed 13,000 rpm or 16,000 g at 4 °C. Carefully remove supernatant of 350 mL by pipetting and transfer it to a new microfuge tube. Use only this supernatant (lysate) in subsequent steps.
  3. Add an equal volume of 70% ethanol into the microfuge tube.
  4. Transfer up to 700 mL of the sample, including any precipitate that may have formed, to a spin column placed in a 2 mL collection tube. Close the lid gently and centrifuge for 15 s at ≥8,000 x g to wash the spin column membrane. Discard the flow-through.
  5. Add 350 mL of buffer RW1 to the spin column. Close the lid gently and centrifuge for 15 s at ≥8,000 x g to wash the spin column membrane. Discard the flow-through and reuse the collection tube in next step.
    CAUTION: Buffer RW1 contains guanidine salts; do NOT mix with bleach.
  6. Add 350 mL of buffer RW1 to the spin column. Close the lid gently and centrifuge for 15 s at ≥8,000 x g. Discard the flow-through.
  7. Add 500 mL of buffer RPE to the spin column. Close the lid gently and centrifuge for 15 s at ≥8,000 x g to wash the spin column membrane. Discard the flow-through.
  8. Add 500 mL of buffer RPE to the spin column. Close the lid gently and centrifuge for 2 min at ≥8,000 x g to wash the spin column membrane. Then carefully remove the spin column from the collection tube, ensuring that the column does not contact the flow-through.
  9. Place the spin column in a new 2 mL collection tube and discard the old tube with the flow-through. Close the lid gently and centrifuge at full speed for 1 min to remove residual buffer.
  10. Place the spin column in a new 1.5 mL collection tube. Add 30-50 mL of RNase-free water directly to the spin column membrane. Close the lid gently and centrifuge for 1 min at ≥8,000 x g to elute the RNA.
  11. If expected RNA yield is >30 mg, repeat step 4.10 with another 30-50 mL of RNase-free water, or using elute from Step 4.10 (if high [RNA] is required). Reuse collection tube from Step 4.10.
  12. Use purified RNA for downstream analysis including RNA-sequencing or real time quantitative PCR or store RNA dissolved in RNase-free H2O at -80 °C for up to 1 year.

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Representative Results

Our previous studies4,7 suggest that CD36 may function as a switch for arteriolar differentiation and capillary arterialization via the LPA/PKD-1 signaling pathway. To study whether the LPA/PKD-1-CD36 signaling axis is essential for arteriogenesis in vivo, we have established the novel TRAP lines that not only have global cd36 deficiency or endothelial-specific-cd36- or pkd-1-deficiency but also permit selective isolation of ribosome-bound RNA from cre-marked cell lineages by GFP, and are useful as a cre-activated fluorescent reporter2

By performing genotyping, we observed that cd36 gene was deleted globally or in the vascular endothelia for endothelial-specific cd36 null mice (data not shown), and pkd-1 gene was also deleted in the vascular endothelia. Figure 1 is a representative result showing the created global cd36 TRAP or endothelial-specific pkd-1 TRAP mouse line. Using immunofluorescence microscopy, we demonstrated that an enhanced GFP is genetically tagged onto the ribosomes of the endothelial cells in vivo (Figure 2). We then isolated ribosome bound mRNA directly in vivo and successfully obtained quality RNA as shown by measurement of the ratio of 260 nm and 230 nm (Figure 3). Further analysis using real-time qPCR demonstrated that the expression of certain arteriogenic genes were upregulated in the lung endothelia of cd36 null mice (Figure 4), indicating that the isolated RNA directly in vivo in the vascular endothelia using the TRAP technology are qualified for downstream studies. These studies include analysis of gene expression at mRNA levels and identification of novel transcriptomes under physiological and pathological conditions, which are essential for understanding the regulation of vascular endothelial cell differentiation and functional angiogenesis. 

Figure 1
Figure 1: An example of genotyping for genetically engineered TRAP mice. Representative results for genotyping of global cd36 null TRAP mice or conditional tissue-specific pkd-1 null TRAP mice. VEC-cre transgenic mice express Cre recombinase under the control of a Cdh5 promoter B6; 129-Tg (Cdh5-cre)1Spe/J mice were bread with B6.129S4-Gt(ROSA)26Sor tm1(CAG-EGFP/Rpl10a,-birA)Wtp/J, and further with B6.129S1tm1Mfe-cd36 /J or pkd-1loxP/loxP. The double mutant cd36 TRAP (A) and pkd-1 TRAP (B) mice were obtained, in which an enhanced GFP is tagged onto L10a of the ribosome in vascular endothelial cells, and cd36 gene is deleted globally and pkd-1 gene specifically in the vascular endothelia. Mouse tails were collected for DNA extraction using a kit and based on the instruction from the manufacturer, and DNA in all samples was amplified by polymerase chain reaction (PCR), and then evaluated by 1-2% agarose-gel electrophoresis. Photographs are the agarose gel image showing the results of amplification of cd36 or pkd-1 mutants with/without TRAP or wild type (WT) mice. Mouse genotype panel A: lane 1, cd36-/-;TRAP+/-; lane 2, TRAP+/+; lane 3, cd36-/-;TRAP+/+;Cdh5+/-; lane 4, TRAP+/+;Cdh5+/-; lane 5, cd36-/-;TRAP+/+;Cdh5+/-; lane 6, cd36-/-;TRAP+/-;Cdh5+/-; lane 7, TRAP+/+;Cdh5+/-; lane 8, TRAP+/-; lane 9, DNA ladder. Mouse genotype panel B: lane 1, pkd-1fl/-; TRAP+/-; Cdh5+/-; lane 2, pkd-1fl/fl; TRAP+/+; Cdh5+/-; lane 3, pkd-1fl/-; TRAP+/+; lane 4, pkd-1fl/fl; TRAP+/+; Cdh5+/-; lane 5, pkd-1fl/fl; TRAP+/+; Cdh5+/-; lane 6, pkd-1fl/-; lane 7, DNA ladder. Please click here to view a larger version of this figure.

Figure 2
Figure 2: An example of endothelial-specific enhanced GFP expression under fluorescence microscope. Blood vascular endothelia in the lung tissues of cd36 knockout TRAP mice were EGFP positive (green color, upper panel) under immunofluorescence microscope. Missing the primary GFP antibody was used as a negative control (bottom panel). Mouse tissues were co-stained by using GFP and CD31 antibodies with appropriate secondary fluorescence antibodies (red color). Representative images acquired by using a fluorescence microscopy imaging system. Bar = 200 µm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: The quality and quantity of ribosomal-bound mRNA of endothelial cells purified and directly extracted from tissues of TRAP mice. An example for quality and concentration of purified RNA from lung tissues in a cd36 knock out TRAP mouse. A spectrophotometer was used for assessment of the amount and purity of extracted RNA. As shown in this figure, the concentration of RNA is 51.2 ng/µL. The ratio of absorbance at 260 nm and 280 nm is 1.87 whereas the ratio of 260 nm and 230 nm is 2.40, indicating the purity of the extracted RNA samples. Please click here to view a larger version of this figure.

Figure 4
Figure 4: An example of expression of angiogenic genes and Notch ligands in the ribosome-bound RNA of endothelial cells by real time qPCR assays. The isolated mRNA from the endothelial ribosome of the lung in the TRAP control and EC-specific cd36 deficient TRAP mice was subjected to real-time qPCR assays, using primers purchased from a biotech company including Hey2, ephrin B2, and delta like ligand 4 (DLL4). The house keeping genes PPIA was used for normalization. The student t-test was used for statistical analysis. *P < 0.05; **P <0.01.

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Discussion

Angiogenesis is a complex multistep process, in which EC-specific angiogenic gene transcription and expression play an essential role in EC differentiation and angiogenic reprogramming3,4. To overcome the barriers from the cellular diversity and architectural complexity for better understanding the function of the mammalian vascular system at a molecular level in vivo, we have created EC-specific TRAP mice, accompanied by EC-specific cd36, EC-specific pkd-1 deficiency or global cd36 deficiency by using a versatile floxed TRAP mouse model or EGFP-TRAP generated in the Pu laboratory2 in combination with other genetically engineered mouse lines. This will allow the examination of the entire translated mRNA complement of vascular ECs from intact tissues in vivo under EC-specific in pkd-1 or global deficiency in cd36 gene expression8, which is critical for investigation into gene transcription associated with physiological and pathological angiogenesis4,7,9,10. Consistent to other studies1,2, our approach to isolation of EC-specific mRNA does not need tissue fixation, dissociation of tissues, or isolation of single-cells from tissues and thus avoids the potential artifacts that result from these treatments. We were also able to perform TRAP purifications and extract quality ribosome-bound mRNA from the frozen tissues. Additionally, what was purified is the translated mRNA content of ECs directly in vivo, which will better represent the protein content compared to using the total RNA for gene expression profile. Moreover, the TRAP transgene genetically labels the ECs with EGFP, also allowing not only for extraction of ribosome-bound mRNA but also for visualization in immunohistochemical or electrophysiological studies. 

However, the approach showed low RNA yields, especially with purified mRNA from heart tissues or from previously frozen tissues. We thus need optimize the conditions to increase yields. However, we observed in EC-specific cd36 deficient mice, the levels of ephrin B2 and DLL4 were significantly increased in both lung (Figure 4) and heart (data not shown) endothelia when compared with the control. These results were consistent with our previous in vitro studies3,4, which suggests that the RNA quality is sufficient for downstream analysis. The yield was low possibly due to the stringent conditions. To overcome this limitation and improve yield, it is critical to set up an RNase-free work zone and decontaminate work surfaces and equipment that may get contaminated with RNase and change gloves frequently in order to extract quality RNA. It is also critical to find suitable concentrations of GFP antibodies in the affinity matrix and use appropriate concentrations of RNase inhibitor in the tissue lysis buffer. Use of RNase-free plastic ware and reagents is beneficial for RNA extraction from endothelial ribosomes of the targeted tissues. 

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Disclosures

The authors declare that they have no conflict of interest.

Acknowledgments

Dr Ren’s work is supported by the American Heart Association (13SDG14800019; BR), the Ann’s Hope Foundation (FP00011709; BR), the American Cancer Society (86-004-26; the MCW Cancer Center to BR), and the National Institute of Health (HL136423; BR); Jordan Palmer is supported by the 2018 MCW CTSI 500 Stars Internship Program; P. Moran is supported by an Institutional Research Training Grant from NHLBI (5T35 HL072483-34). 

Materials

Name Company Catalog Number Comments
2100 Electrophoresis Bioanalyzer with Nanochips and Picochips Agilent G2939AA, 5067-1511 & 5067-1513
Cell scrapers Sarstedt 83.1832
Homogenizers Fisher Scientific K8855100020
Magnet (Dynamag-2) Invitrogen 123-21D Will depend on purification scale; samples in 1.5-mL tubes can be concentrated on a DynaMag-2
Minicentrifuge Fisher Scientific 05-090-100
NanoDrop 2000C spectrophotometer Thermo Scientific  ND-2000C
Refrigerated centrifuge Eppendorf 5430R with rotor for 1.5-mL microcentrifuge tubes
RNase-free 1.5mL microcentrifuge tubes Applied Biosystems  AM12450
Rnase-free 50-mL conical tubes Applied Biosystems  AM12501
RNase-free 1000-μl filter tips Rainin RT-1000F
RNase-free 200-μl filter tips Rainin  RT-200F
RNase-free 20-μl filter tips Rainin  RT-20F
Rotor for homogenizers Yamato  LT-400D
Tube rotator, Labquake brand Thermo Fisher 13-687-12Q

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References

  1. Heiman, M., Kulicke, R., Fenster, R. J., Greengard, P., Heintz, N. Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nature Protocols. 9, 1282-1291 (2014).
  2. Zhou, P., et al. Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification. Proceedings of the National Academy of Sciences of the United States of America. 110, 15395-15400 (2013).
  3. Best, B., Moran, P., Ren, B. VEGF/PKD-1 signaling mediates arteriogenic gene expression and angiogenic responses in reversible human microvascular endothelial cells with extended lifespan. Molecular and Cellular Biochemistry. 446, 199-207 (2018).
  4. Ren, B., et al. LPA/PKD-1-FoxO1 Signaling Axis Mediates Endothelial Cell CD36 Transcriptional Repression and Proangiogenic and Proarteriogenic Reprogramming. Arteriosclerosclerosis Thrombosis, Vascular Biology. 36, 1197-1208 (2016).
  5. Ren, B. Protein Kinase D1 Signaling in Angiogenic Gene Expression and VEGF-Mediated Angiogenesis. Frontiers in Cell and Developmental Biology. 4, 37 (2016).
  6. Ren, B. FoxO1 transcriptional activities in VEGF expression and beyond: a key regulator in functional angiogenesis? Journal of Pathology. 245, 255-257 (2018).
  7. Hupe, M., Li, M. X., Gertow Gillner, K., Adams, R. H., Stenman, J. M. Evaluation of TRAP-sequencing technology with a versatile conditional mouse model. Nucleic Acids Research. 42, e14 (2014).
  8. Dong, L., et al. Diet-induced obesity links to ER positive breast cancer progression via LPA/PKD-1-CD36 signaling-mediated microvascular remodeling. Oncotarget. 8, 22550-22562 (2017).
  9. Ren, B., et al. ERK1/2-Akt1 crosstalk regulates arteriogenesis in mice and zebrafish. Journal of Clinical Investigation. 120, 1217-1228 (2010).
  10. Skuli, N., et al. Endothelial HIF-2alpha regulates murine pathological angiogenesis and revascularization processes. Journal of Clinical Investigation. 122, 1427-1443 (2012).

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