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In This Article

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

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

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.

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Disclosures

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

NameCompanyCatalog NumberComments
PNA probePANAGENEcustom order
Anti-Digoxigenin-Fluorescein, Fab fragmentsRoche11207741910use 1:200 diluted in PBST
Digoxigenin-dUTPRoche11573152910
Bovine serum albuminSIGMA-ALDRICHA-7906
ParaformaldehydeSIGMA-ALDRICHP-6148prepare 4% paraformaldehyde by heating in DW with few drops of NaOH. add 0.1 volume of 10x PBS.
VectashieldVector LaboratoriesH-1200
Hybridizaiton solution3X 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 solution2X SSC, 50% formamide
FormamideBIONEERC-9012toxic
MethanolCarlo Erba
AcetoneCarlo Erba
HeparinSIGMA-ALDRICHH3393make 10 mg/ml for stock solution
Dextran sulfateSIGMA-ALDRICH67578
10X PBSFor 1 Liter DW : 80 g NaCl, 2.0 g KCl, 27 g Na2HPO4:7H2O, 2.4 g KH2PO
PBST1X PBS, 0.1% tween-20
Polysorbate 20SIGMA-ALDRICHP-2287Commercial name is Tween-20
Poly-L-Lysine solution (0.1 % w/v)SIGMA-ALDRICHP-8920prepare fresh 0.01 % w/v solution before use
M93 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L
Bleaching solution20% sodium hypochlorite, 0.5 M KOH
Antibody buffer1X PBST, 1mM EDTA, 0.1% BSA, 0.05% Sodium azide (toxic)
Blocking solutionAntibody buffer with 5% bovine serum albumin (BSA)
illustra Microspin G-50GE healthcare27-53310-01
20X SSCTo make 1L, 175.3 g of NaCl, 88.2 g of sodium citrate, H2O to 1 L, adjust pH to 7.0
2X SSCT2X SSC, 0.1 % tween-20
10x digoxigenin-dUTP mix1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP
PCR purification columnsCosmo genetechCMR0112
Glass cleaner / ULTRA CLEANDukssan pure chemicals8AV721
Multi-well glass slideMP biomedicals96041205
Nematode growth mediato 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.
LevamisoleSIGMA-ALDRICH196142
RazorFeatherblade No. 11
Rnase AEnzynomics
BSASIGMA-ALDRICHA7906
Equipments
Confocal microsopeZeissLSM 510EC Plan-Neofluar 100x was used as objective lens.
Dry block / aluminum blockLabtechLBH-T03Set temperature to 80℃
Humid chamberPlastic box filled with paper towel soaked in DW
Image Analysis Software Dr. Peter LandsdorpTFL-telohttp://www.flintbox.com/public/project/502

References

  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 ....

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