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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.
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.
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 ....
1 . Selection of Neomycin Resistant Clones
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.......
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.......
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).
....Name | Company | Catalog Number | Comments |
AGS cells | - | - | Gift from Christian Baron (Université de Montréal). |
F12K Nut Mix 1X | GIBCO | 21127022 | Culturing medium for AGS cells |
L-Glutamine | CORNING | MT25005CI | Component of cell culturing medium |
Penicillin Streptomycin Solution | CORNING | 30-002-CI | Component of cell culturing medium |
Fetal Bovine Serum | Sigma Aldrich | F2442 | Component of cell culturing medium |
DMEM 1X | GIBCO | 21068028 | culturing medium for phoenix cell |
CaCl2 | Sigma Aldrich | C1016 | used in phoenix cell transfection |
HEPES | Sigma Aldrich | H3375 | used in phoenix cell transfection (HBS solution) |
KCl | Sigma Aldrich | P9333 | used in phoenix cell transfection (HBS solution) |
Dextrose | Sigma Aldrich | D9434 | used in phoenix cell transfection (HBS solution) |
NaCl | Sigma Aldrich | S7653 | used in phoenix cell transfection (HBS solution) and retrovirus precipitation |
Na2HPO4 | Sigma Aldrich | S3264 | used in phoenix cell transfection (HBS solution) |
TRYPSIN EDTA SOLUTION 1X | CORNING | 59430C | used in cell split |
DPBS 1X | GIBCO | 14190250 | Dulbecco's Phosphate Buffered Saline |
DMSO | Sigma Aldrich | D8418 | Component of cell freezing medium (80% FBB and 20% DMSO) |
G-418 Disulphate | Formedium | G4185 | selection drug for |
Gelatin solution Bioreagent | Sigma Aldrich | G1393 | cotaing of 96 well DNA plate and freezing plate |
Tris-base | Fisher BioReagents | 10376743 | Component of Cell lysis buffer for genomic DNA extraction |
EDTA | Sigma Aldrich | E6758 | Component of Cell lysis buffer for genomic DNA extraction |
SDS | Sigma Aldrich | 71729 | Component of Cell lysis buffer for genomic DNA extraction |
Proteinase K | Thermo Fisher | AM2546 | Component of Cell lysis buffer for genomic DNA extraction |
RNAse A | Thermo Fisher | 12091021 | RNA degradation during DNA extraction |
Agarose | Sigma Aldrich | A5304 | DNA gel preparation |
Atlas ClearSight | Bioatlas | BH40501 | Stain reagent used for detecting DNA and RNA samples in agarose gel |
ethanol | Fisher BioReagents | BP28184 | DNA precipitation |
Sodium Acetate | Sigma Aldrich | 71196 | Used for DNA precipitation at a 3M concentration pH5.2 |
Wizard SV Gel and PCR clean-Up system | Promega | A9282 | Extraction of PCR fragments from agarose gel during PCR screening of neomycin positive clones |
Trizol | AMBION | 15596018 | Organic solvent used for RNA extraction |
Dnase I | THERMO SCIENTIFIC | 89836 | degradation of genomic DNA from RNAÂ |
dNTPs mix | Invitrogen | 10297018 | used in RT and PCR reactions |
DTT | Invitrogen | 707265ML | used in RT reactions |
diethyl pyrocarbonate | Sigma Aldrich | D5758 | used to inactivate RNAses in water (1:1000 dilution) |
Ribolock | Thermo Fisher | EO0381 | RNase inhibitor |
MOPS | Sigma Aldrich | M9381 | preparation of RNA gel |
Paraformaldehyde | Electron Microscopy Sciences | 15710 | preparation of denaturating RNA gel (1% PFA in 1x MOPS) |
Superscript III Reverse transcriptase | Invitrogen | 18080-093 | Retrotranscription reaction |
Pfu DNA polymerase (recombinant) | Thermo Scientific | EP0501 | PCR reaction |
2X qPCRBIO SyGreen Mix Separate-ROX | PCR BIOSYSTEMS | PB 20.14 | qPCR reaction |
Cre-GFP adenovirus | https://medicine.uiowa.edu/vectorcore | 1174-HT | used to infect TERRA-MS2 clones in order to remove the neomycn gene |
Sodium Butyrate | Sigma Aldrich | B5887 | used to promote retrovirus particles production in phoenix cells |
PEG8000 | Sigma Aldrich | 89510 | Precipitation of retrovirus partcles |
35µ-Dish Glass Bottom | Ibidi | 81158 | used in live cell imaging analyses of TERRA-MS2 clones |
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