端粒功能失调诱导病灶（TIF）分析

Ilgen Mender; Jerry W. Shay

doi:10.21769/BioProtoc.1656

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Peer-reviewed

Telomere Dysfunction Induced Foci (TIF) Analysis

端粒功能失调诱导病灶（TIF）分析

Ilgen Mender

Jerry W. Shay email

发布: 2015年11月20日第5卷第22期 DOI: 10.21769/BioProtoc.1656 浏览次数: 12242

评审: HongLok LungVanesa Olivares-IllanaAnonymous reviewer(s)

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Cover of Cancer Discovery, featuring study using the protocol.

Jan 2015

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Abstract

Telomerase maintains telomeric DNA in eukaryotes during early developments, ~90% of cancer cells and some proliferative stem like cells. Telomeric repeats at the end of chromosomes are associated with the shelterin complex. This complex consists of TRF1, TRF2, Rap1, TIN2, TPP1, POT1 which protect DNA from being recognized as DNA double-stranded breaks. Critically short telomeres or impaired shelterin proteins can cause telomere dysfunction, which eventually induces DNA damage responses at the telomeres. DNA damage responses can be identified by antibodies to 53BP1, gammaH2AX, Rad17, ATM, and Mre11. DNA damage foci at uncapped telomeres are referred to as Telomere dysfunction-Induced Foci (TIFs) (de Lange, 2005; Takai et al., 2003).

The TIF assay is based on the co-localization detection of DNA damage by an antibody against DNA damage markers, such as gamma-H2AX, and telomeres using an antibody against one of the shelterin proteins such as TRF2 (Takai et al., 2003; de Lange, 2002; Karlseder et al., 1999). The method we describe here can be used in normal human and cancer cells.

Other commonly used methods-Telomere Restriction Fragment (TRF) Analysis (Mender and Shay, 2015b) and Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015a)- in telomere biology can be found by clicking on the indicated links.

Keywords: Telomere dysfunction induced foci (端粒功能障碍致病灶)

Telomeres (端粒酶)

Telomerase (端粒酶)

Telomeric DNA damage (端粒DNA损伤)

Materials and Reagents

Lab-Tek II Chamber slides (Thomas Scientific, catalog number: 154461 )
Cancer or normal human cells
Triton X-100 (J.T. Baker Chemical Co., catalog number: 7-x198 )
Octyl Phenol Ethoxylate (Avantor Performance Materials, J.T. Baker, catalog number: X198-07 )
Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: P6148 )
Nonidet-P40 (Fluka BioChemika, catalog number: 74385 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 74385”.
Fish gelatin blocking buffer, 10% (Amresco, catalog number: M319 )
Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P )
Anti-phospho-histone H2A.X (Ser139, mouse) Antibody (Merck Millipore Corporation, catalog number: 05-636 )
Anti-TRF2 Antibody [EPR3517 (Ouellette et al., 2000)] (rabbit) (Abcam, catalog number: ab108997 )
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor^® 488 conjugate (Invitrogen, catalog number: A-21206 )
Note: Currently, it is “Thermo Fisher Scientific, Novex^TM, catalog number: A-21206”.
Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor^® 568 conjugate (Invitrogen, catalog number: A-11004 )
Note: Currently, it is “Thermo Fisher Scientific, Novex^TM, catalog number: A-11004”.
Vectashield^® Mounting Medium with DAPI (Vector Laboratories, catalog number: H-1200 )
Deltavision^® Immersion Oil N=1.518 (GEHC, part no: 290291717 )
Phosphate-buffered saline (PBS)
MilliQ^® water
10x PBS buffer-phosphate buffer saline (see Recipes)
1x PBST buffer (see Recipes)

Equipment

Personal DeltaVision wide-field fluorescent microscope (GE Healthcare, model: PD11435 )
Lamp (Xenon)
Camera (Photometrics)
60x/1.42 N.A. (numerical aperture) objective (Olympus)

Software