JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Published: August 4th, 2016

DOI:

10.3791/54224

1Institute of Molecular Biology and Genetics (IMBG), Seoul National University, 2Department of Biological Sciences, Seoul National University, 3Department of Biophysics and Chemical Biology, Seoul National University

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).

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.

Telomere protects chromosomal ends from aberrant fusion and degradation. Mammalian telomere is composed of G-rich hexameric repeats, TTAGGG, and shelterin complexes. The telomere repeat sequence of the nematode is similar to those of mammals (TTAGGC). Most eukaryotes utilize telomerase to add telomere repeats to their chromosomal ends. However, 10 - 15% of cancer cells utilize telomerase independent mechanism, known as Alternative Lengthening of Telomeres (ALT)3. Previously, we reported that telomere repeats and its associated sequences, named as TALT, were amplified in the telomeres of telomerase mutant lines that survived critical sterility2.

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Labeling Probes with Digoxigenin-dUTP by PCR

  1. Perform PCR labeling with 10x dNTP mix containing digoxigenin-dUTP as previously described13.
  2. Purify PCR product with spin-column purification according to manufacturer's instruction.
    1. If the probe is shorter than 200 bp, remove free digoxigenin-dUTP with spin-column chromatography from the reaction mixture rather than spin-column purification.

2. Preparing Polylysine Coated Slide.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

It was previously reported that ALT survivor can emerge from telomerase-deficient mutant, trt-1(ok410), in low frequency by replicating internally localized 'Template of ALT' (TALT) sequences for telomere maintenance2. Using PNA probe, we were able to visualize telomeres in the dissected gonads (Figure 2A). The faint telomere signal was detected both in trt-1(ok410) and ALT survivor. The fuzzy signal was overlapped only with DAPI, sugg.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The main advantage of our protocol is the simplicity of the procedure without noticeable damage to the morphology of cellular structure. Several steps were optimized for C. elegans FISH in this protocol. The critical steps for successful FISH include labeling of probes, fixation of embryos and penetration. Digoxigenin-dUTP labeling method provides an easy-to-use labeling method by PCR or nick-translation. To label long target sequence, nick-translation is preferred. In this case, the probes should be digested wi.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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).

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
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

  1. Reddel, R. R., Bryan, T. M., Murnane, J. P. Immortalized cells with no detectable telomerase activity. A review. Biochemistry-Moscow. 62, 1254-1262 (1997).
  2. Seo, B., et al. Telomere maintenance through recruitment of internal genomic regions. Nat Commun. 6, 8189 (2015).
  3. Cesare, A. J., Reddel, R. R. Alternative lengthening of telomeres: models, mechanisms and implications. Nat Rev Genet. 11, 319-330 (2010).
  4. Meier, B., et al. trt-1 is the Caenorhabditis elegans catalytic subunit of telomerase. Plos Genetics. 2, 187-197 (2006).
  5. Cawthon, R. M. Telomere measurement by quantitative PCR. Nucleic Acids Res. 30, 47 (2002).
  6. Raices, M., Maruyama, H., Dillin, A., Karlseder, J. Uncoupling of longevity and telomere length in C. elegans. PLoS Genet. 1, 30 (2005).
  7. Southern, E. M. Detection of Specific Sequences among DNA Fragments Separated by Gel-Electrophoresis. Journal of Molecular Biology. 98, 503 (1975).
  8. Lee, M., et al. Telomere extension by telomerase and ALT generates variant repeats by mechanistically distinct processes. Nucleic Acids Res. 42, 1733-1746 (2014).
  9. Cheung, I., et al. Strain-specific telomere length revealed by single telomere length analysis in Caenorhabditis elegans. Nucleic Acids Res. 32, 3383-3391 (2004).
  10. Shtessel, L., et al. Caenorhabditis elegans POT-1 and POT-2 repress telomere maintenance pathways. G3. 3, 305-313 (2013).
  11. Duerr, J. Immunohistochemistry. WormBook (The C. elegans Research Community). , (2006).
  12. Phillips, C. M., McDonald, K. L., Dernburg, A. F. Cytological analysis of meiosis in Caenorhabditis elegans. Meiosis: Volume 2, Cytological Methods. , 171-195 (2009).
  13. Emanuel, J. R. Simple and efficient system for synthesis of non-radioactive nucleic acid hybridization probes using PCR. Nucleic acids research. 19, 2790 (1991).
  14. Stiernagle, T. Maintenance of C. elegans. WormBook. , 1-11 (2006).
  15. Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Ceron, J. Basic Caenorhabditis elegans methods: synchronization and observation. J Vis Exp. , e4019 (2012).
  16. 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).
  17. 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).
  18. Lee, M. H., Schedl, T. RNA in situ hybridization of dissected gonads. WormBook. , 1-7 (2006).
  19. 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).
  20. Poon, S. S., Lansdorp, P. M. Quantitative fluorescence in situ hybridization (Q-FISH). Curr Protoc Cell Biol. 18, 14 (2001).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved