Observation og Kvantificering af telomer og repetitive sekvenser Brug Fluorescens<em> In Situ</em> Hybridisering (FISH) med PNA-prober i<em> Caenorhabditis elegans</em
Summary
We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).
Abstract
Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.
Introduction
Telomer beskytter kromosomale ender fra afvigende fusion og nedbrydning. Mammal telomer består af G-rige hexamere gentagelser, TTAGGG og shelterin komplekser. Den telomer gentagelsessekvens af nematoden ligner dem af pattedyr (TTAGGC). De fleste eukaryoter udnytte telomerase at tilføje telomer gentagelser til deres kromosomale ender. Men, 10 – 15% af kræftceller udnytte telomerase uafhængig mekanisme, kendt som Alternativ Forlængelse af Telomerer (ALT) 3. Tidligere vi rapporterede, at telomer gentagelser og dens tilhørende sekvenser, navngivet som Tält, blev opformeret i telomerer af telomerase mutant linjer, der overlevede kritisk sterilitet 2.
Telomerlængde blev målt ved kvantitativ PCR eller ved Southern blot, som tilvejebringer gennemsnitlige længde af de samlede telomerer 4,5,6,7. Læs optælling af telomer repeat i hele genomet sekventering data er også en indikator af de samlede telomer indhold 8. selvom SinGLE telomerlængde Analysis (STELA) kunne give længden af en enkelt telomer, kan det ikke give geografisk information af telomerer 9. Mens POT-1 :: mCherry reporter protein giver den geografisk information af telomerer in vivo, kan det ikke repræsentere længder af dobbelt-strenget telomerer, som POT-1 er en enkelt-strenget telomer bindende protein 10.
Mens ovennævnte fremgangsmåder giver det gennemsnitlige information af repetitive sekvenser, fluorescens in situ hybridisering (FISH) gør det muligt at observere mængden og rumlige mønster af individuelle sekvenser af interesse på en kromosomal skala. I stedet for rensning af DNA, er væv eller celler fikseret for at bevare den indfødte geografisk information i FISH. , FISH er således en både kvantitativ og kvalitativ redskab til observation af individuelle repeat sekvenser, såsom telomer gentagelser.
Denne protokol giver en effektiv fremgangsmåde til samtidig detektion af både teloblotte og andre gentagelser baseret på forbedringer fra tidligere beskrevne metoder 11,12. C. elegans larver eller voksne er flercellede organisme med højt differentierede celler. Heterogenitet celler hæmmer på kvantitativ analyse af et stort antal telomer pletter. For at maksimere antallet af analyserede celler, embryoer er isoleret og spredt på polylysin-belagte dias for FISH. Derudover kan denne protokol også kombineres med immunofluorescens.
Som bevis for, at protokollen fungerer, viser vi, at det er muligt at observere og kvantificere to forskellige repetitive sekvenser. DNA-probe mod TALT1 blev genereret med enkle PCR indarbejde digoxigenin-dUTP. Så denne TALT1 probe og fluorescensmærket telomer PNA probe blev hybridiseret samtidigt. Efterfølgende digoxigenin blev påvist ved kanoniske immunofluorescens metoder. Vi præsenterer her de repræsentative billeder, hvor TALT1 colocalized med telomer i TRT-1 </em> overlevende.
Protocol
Representative Results
Discussion
Den største fordel ved vores protokol er enkeltheden af proceduren uden mærkbar skade på morfologien af cellestrukturen. Adskillige trin blev optimeret til C. elegans FISH i denne protokol. De kritiske trin for en vellykket FISH omfatter mærkning af sonder, fiksering af embryoner og penetration. Digoxigenin-dUTP mærkning metode giver en nem-at-bruge metoden ved PCR eller nick-translation mærkning. For at mærke lang målsekvens, er nick-translation foretrækkes. I dette tilfælde bør proberne…
Declarações
The authors have nothing to disclose.
Acknowledgements
Mutant worm strains were kindly provided by the Caenorhabditis Genetics Center. This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI14C1277).
Materials
| PNA probe | PANAGENE | custom order | |
| Anti-Digoxigenin-Fluorescein, Fab fragments | Roche | 11207741910 | use 1:200 diluted in PBST |
| Digoxigenin-dUTP | Roche | 11573152910 | |
| Bovine serum albumin | SIGMA-ALDRICH | A-7906 | |
| Paraformaldehyde | SIGMA-ALDRICH | P-6148 | prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS. |
| Vectashield | Vector Laboratories | H-1200 | |
| Hybridizaiton solution | 3X SSC, 50% formamide, 10% (w/v) dextran sulfate, 50 ug/ml heparin, 100 ug/ml yeast tRNA , 100ug/ml sonicated salmon sperm DNA | ||
| Hybridizaiton wash solution | 2X SSC, 50% formamide | ||
| Formamide | BIONEER | C-9012 | toxic |
| Methanol | Carlo Erba | ||
| Acetone | Carlo Erba | ||
| Heparin | SIGMA-ALDRICH | H3393 | make 10 mg/ml for stock solution |
| Dextran sulfate | SIGMA-ALDRICH | 67578 | |
| 10X PBS | For 1 Liter DW : 80 g NaCl, 2.0 g KCl, 27 g Na2HPO4:7H2O, 2.4 g KH2PO | ||
| PBST | 1X PBS, 0.1% tween-20 | ||
| Polysorbate 20 | SIGMA-ALDRICH | P-2287 | Commercial name is Tween-20 |
| Poly-L-Lysine solution (0.1 % w/v) | SIGMA-ALDRICH | P-8920 | prepare fresh 0.01 % w/v solution before use |
| M9 | 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L | ||
| Bleaching solution | 20% sodium hypochlorite, 0.5 M KOH | ||
| Antibody buffer | 1X PBST, 1mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic) | ||
| Blocking solution | Antibody buffer with 5% bovine serum albumin (BSA) | ||
| illustra Microspin G-50 | GE healthcare | 27-53310-01 | |
| 20X SSC | To make 1L, 175.3 g of NaCl, 88.2 g of sodium citrate, H2O to 1 L, adjust pH to 7.0 | ||
| 2X SSCT | 2X SSC, 0.1 % tween-20 | ||
| 10x digoxigenin-dUTP mix | 1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP | ||
| PCR purification columns | Cosmo genetech | CMR0112 | |
| Glass cleaner / ULTRA CLEAN | Dukssan pure chemicals | 8AV721 | |
| Multi-well glass slide | MP biomedicals | 96041205 | |
| Nematode growth media | to make 1 L, 3 g of NaCl, 17 g of agar, 2.5 g of peptone, H2O to 974 mL. Autoclave and cool the flask. Add 1 mL of 1M CaCl2, 1 ml of 4 mg/mL cholesterol in ethanol, 1 ml of 1 M MgSO4, 25 mL of 1 M KPO4. | ||
| Levamisole | SIGMA-ALDRICH | 196142 | |
| Razor | Feather | blade No. 11 | |
| Rnase A | Enzynomics | ||
| BSA | SIGMA-ALDRICH | A7906 | |
| Equipments | |||
| Confocal microsope | Zeiss | LSM 510 | EC Plan-Neofluar 100x was used as objective lens. |
| Dry block / aluminum block | Labtech | LBH-T03 | Set temperature to 80℃ |
| Humid chamber | Plastic box filled with paper towel soaked in DW | ||
| Image Analysis Software | Dr. Peter Landsdorp | TFL-telo | http://www.flintbox.com/public/project/502 |
Referências
- Reddel, R. R., Bryan, T. M., Murnane, J. P. Immortalized cells with no detectable telomerase activity. A review. Biochemistry-Moscow. 62, 1254-1262 (1997).
- Seo, B., et al. Telomere maintenance through recruitment of internal genomic regions. Nat Commun. 6, 8189 (2015).
- Cesare, A. J., Reddel, R. R. Alternative lengthening of telomeres: models, mechanisms and implications. Nat Rev Genet. 11, 319-330 (2010).
- Meier, B., et al. trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase. Plos Genetics. 2, 187-197 (2006).
- Cawthon, R. M. Telomere measurement by quantitative PCR. Nucleic Acids Res. 30, 47 (2002).
- Raices, M., Maruyama, H., Dillin, A., Karlseder, J. Uncoupling of longevity and telomere length in C. elegans. PLoS Genet. 1, 30 (2005).
- Southern, E. M. Detection of Specific Sequences among DNA Fragments Separated by Gel-Electrophoresis. Journal of Molecular Biology. 98, 503 (1975).
- Lee, M., et al. Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes. Nucleic Acids Res. 42, 1733-1746 (2014).
- Cheung, I., et al. Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans. Nucleic Acids Res. 32, 3383-3391 (2004).
- Shtessel, L., et al. Caenorhabditis elegans POT-1 and POT-2 repress telomere maintenance pathways. G3. 3, 305-313 (2013).
- Duerr, J. Immunohistochemistry. WormBook (The C. elegans Research Community). , (2006).
- Phillips, C. M., McDonald, K. L., Dernburg, A. F. Cytological analysis of meiosis in Caenorhabditis elegans. Meiosis: Volume 2, Cytological Methods. , 171-195 (2009).
- Emanuel, J. R. Simple and efficient system for synthesis of non-radioactive nucleic acid hybridization probes using PCR. Nucleic acids research. 19, 2790 (1991).
- Stiernagle, T. Maintenance of C. elegans. WormBook. , 1-11 (2006).
- Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Ceron, J. Basic Caenorhabditis elegans methods: synchronization and observation. J Vis Exp. , e4019 (2012).
- Poon, S. S. S., Martens, U. M., Ward, R. K., Lansdorp, P. M. Telomere length measurements using digital fluorescence microscopy. Cytometry. 36, 267-278 (1999).
- Ferreira, H. C., Towbin, B. D., Jegou, T., Gasser, S. M. The shelterin protein POT-1 anchors Caenorhabditis elegans telomeres through SUN-1 at the nuclear periphery. J Cell Biol. 203, 727-735 (2013).
- Lee, M. H., Schedl, T. RNA in situ hybridization of dissected gonads. WormBook. , 1-7 (2006).
- Tabara, H., Motohashi, T., Kohara, Y. A multi-well version of in situ hybridization on whole mount embryos of Caenorhabditis elegans. Nucleic Acids Res. 24, 2119-2124 (1996).
- Poon, S. S., Lansdorp, P. M. Quantitative fluorescence in situ hybridization (Q-FISH). Curr Protoc Cell Biol. 18, 14 (2001).
