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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Abstract

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Introduction

The long noncoding RNA TERRA is transcribed from the subtelomeric region of chromosomes and its transcription proceeds towards the chromosome ends, terminating within the telomeric repeat tract1,2. For this reason, TERRA transcripts consist of subtelomeric-derived sequences at their 5' end and terminate with telomeric repeats (UUAGGG in vertebrates)3. Important roles have been proposed for TERRA, including heterochromatin formation at telomeres4,5, DNA replication6, promoting homologous recombination ....

Protocol

1 . Selection of Neomycin Resistant Clones

  1. Grow AGS cells in Ham's F-12K (Kaighn's) medium supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, penicillin (0.5 units per mL of medium), and streptomycin (0.2 µg per mL of medium) at 37 °C and 5% CO2. Transfect the cells at a 50-60% confluence with the sgRNA/Cas9 expressing vector and the MS2 cassette at a 1:10 molar ratio21.
    NOTE: In a parallel experiment, verify transfection efficiency by.......

Representative Results

Figure 1 represents an overview of the experimental strategy. The main steps of the protocol and an indicative timeline for the generation of TERRA-MS2 clones in AGS cells are shown (Figure 1A). At day 1, multiple wells of a 6 well plate are transfected with the MS2 cassette and sgRNA/Cas9 expressing vectors (shown in Figure 1B). Two different subtelomere.......

Discussion

In this article we present a method to generate human cancer cell clones containing MS2 sequences integrated within subtelomere 15q. Using these clones, the MS2-tagged TERRA molecules transcribed from the subtelomere 15q are detected by fluorescence microscopy by co-expression of a MS2-GFP fusion protein. This approach enables researchers to study the dynamics of TERRA expressed from a single telomere in living cells21. In this protocol, TERRA-MS2 clones are selected in the AGS cell line, which re.......

Acknowledgements

We are grateful to the staff of the advanced imaging facility of CIBIO at the University of Trento and the BioOptics Light Microscopy facility at the Max F. Perutz Laboratories (MFPL) in Vienna. The research leading to these results has received funding from the Mahlke-Obermann Stiftung and the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no 609431 to EC. EC is supported by a Rita Levi Montalcini fellowship from the Italian Ministry of Education University and Research (MIUR).

....

Materials

NameCompanyCatalog NumberComments
AGS cells--Gift from Christian Baron (Université de Montréal).
F12K Nut Mix 1XGIBCO21127022Culturing medium for AGS cells
L-Glutamine CORNINGMT25005CIComponent of cell culturing medium
Penicillin Streptomycin SolutionCORNING30-002-CIComponent of cell culturing medium
Fetal Bovine SerumSigma AldrichF2442Component of cell culturing medium
DMEM 1XGIBCO21068028culturing medium for phoenix cell
CaCl2Sigma AldrichC1016used in phoenix cell transfection
HEPESSigma AldrichH3375used in phoenix cell transfection (HBS solution)
KClSigma AldrichP9333used in phoenix cell transfection (HBS solution)
DextroseSigma AldrichD9434used in phoenix cell transfection (HBS solution)
NaClSigma AldrichS7653used in phoenix cell transfection (HBS solution) and retrovirus precipitation
Na2HPO4Sigma AldrichS3264used in phoenix cell transfection (HBS solution)
TRYPSIN EDTA SOLUTION 1XCORNING59430Cused in cell split
DPBS 1XGIBCO14190250Dulbecco's Phosphate Buffered Saline
DMSOSigma AldrichD8418Component of cell freezing medium (80% FBB and 20% DMSO)
G-418 DisulphateFormediumG4185selection drug for 
Gelatin solution BioreagentSigma AldrichG1393cotaing of 96 well DNA plate and freezing plate
Tris-baseFisher BioReagents10376743Component of Cell lysis buffer for genomic DNA extraction
EDTASigma AldrichE6758Component of Cell lysis buffer for genomic DNA extraction
SDSSigma Aldrich71729Component of Cell lysis buffer for genomic DNA extraction
Proteinase KThermo FisherAM2546Component of Cell lysis buffer for genomic DNA extraction
RNAse AThermo Fisher12091021RNA degradation during DNA extraction
AgaroseSigma AldrichA5304DNA gel preparation
Atlas ClearSightBioatlasBH40501Stain reagent used for detecting DNA and RNA samples in agarose gel
ethanolFisher BioReagentsBP28184DNA precipitation
Sodium Acetate Sigma Aldrich71196Used for DNA precipitation at a 3M concentration pH5.2
Wizard SV Gel and PCR clean-Up systemPromegaA9282Extraction of PCR fragments from agarose gel during PCR screening of neomycin positive clones
TrizolAMBION15596018Organic solvent used for RNA extraction
Dnase ITHERMO SCIENTIFIC89836degradation of genomic DNA from RNA 
dNTPs mixInvitrogen10297018used in RT and PCR reactions
DTTInvitrogen707265MLused in RT reactions
diethyl pyrocarbonateSigma AldrichD5758used to inactivate RNAses in water (1:1000 dilution)
RibolockThermo FisherEO0381RNase inhibitor
MOPSSigma AldrichM9381preparation of RNA gel
ParaformaldehydeElectron Microscopy Sciences15710preparation of denaturating RNA gel (1% PFA in 1x MOPS)
Superscript III Reverse transcriptaseInvitrogen18080-093Retrotranscription reaction
Pfu DNA polymerase (recombinant)Thermo ScientificEP0501PCR reaction
2X qPCRBIO SyGreen Mix Separate-ROXPCR BIOSYSTEMSPB 20.14qPCR reaction
Cre-GFP adenovirushttps://medicine.uiowa.edu/vectorcore1174-HTused to infect TERRA-MS2 clones in order to remove the neomycn gene
Sodium ButyrateSigma AldrichB5887used to promote retrovirus particles production in phoenix cells
PEG8000Sigma Aldrich89510Precipitation of retrovirus partcles
35µ-Dish Glass BottomIbidi81158used in live cell imaging analyses of TERRA-MS2 clones

References

  1. Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E., Lingner, J. Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science. 318 (5851), 798-801 (2007).
  2. Schoeftner, S., Blasco, M. A.

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