Combining RNAi and in vivo confocal microscopy analysis of the photoconvertible fluorescent protein Dendra2 to study a DNA repair protein

Gianluca Tell, Matteo Di Piazza, Malgorzata Kamocka, Carlo Vascotto

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Clinical approaches for tumor treatment often rely on combination therapy where a DNA damaging agent is used in combination with a DNA repair protein inhibitor. For this reason, great efforts have been made during the last decade to identify inhibitors of DNA repair proteins or, alternatively, small molecules that specifically alter protein stability or trafficking. Unfortunately, when studying these drug candidates, classical biochemical approaches are prone to artifacts. The apurinic/apyrimidinic endonuclease (APE1) protein is an essential component of the base excision repair (BER) pathway that is responsible for repairing DNA damage caused by oxidative and alkylating agents. In this work, we combined conditional gene expression knockdown of APE1 protein by RNA interference (RNAi) technology with re-expression of an ectopic recombinant form of APE1 fused with the photoconvertible fluorescent protein (PCFP) Dendra2. Dendra2 did not alter the subcellular localization or endonuclease activity of APE1. We calculated APE1 half-life and compared these results with the classical biochemical approach, which is based on cycloheximide (CHX) treatment. In conclusion, we combined RNAi and in vivo confocal microscopy to study a DNA repair protein demonstrating the feasibility and the advantage of this approach for the study of the cellular dynamic of a DNA repair protein.

Original languageEnglish
Pages (from-to)198-203
Number of pages6
JournalBioTechniques
Volume55
Issue number4
DOIs
StatePublished - Oct 2013

Fingerprint

Confocal microscopy
RNA Interference
Confocal Microscopy
DNA Repair
Repair
RNA
DNA
Proteins
Endonucleases
DNA-(Apurinic or Apyrimidinic Site) Lyase
Gene Knockdown Techniques
Protein Stability
Alkylating Agents
Protein Transport
Cycloheximide
Intravital Microscopy
Genetic Therapy
Artifacts
DNA Damage
Half-Life

Keywords

  • APE1/Ref-1
  • Dendra2
  • Live confocal microscopy
  • Protein half-life
  • RNAi

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biotechnology

Cite this

Combining RNAi and in vivo confocal microscopy analysis of the photoconvertible fluorescent protein Dendra2 to study a DNA repair protein. / Tell, Gianluca; Di Piazza, Matteo; Kamocka, Malgorzata; Vascotto, Carlo.

In: BioTechniques, Vol. 55, No. 4, 10.2013, p. 198-203.

Research output: Contribution to journalArticle

@article{199bc3bb6ccd4213b59c66ba8b5ddafe,
title = "Combining RNAi and in vivo confocal microscopy analysis of the photoconvertible fluorescent protein Dendra2 to study a DNA repair protein",
abstract = "Clinical approaches for tumor treatment often rely on combination therapy where a DNA damaging agent is used in combination with a DNA repair protein inhibitor. For this reason, great efforts have been made during the last decade to identify inhibitors of DNA repair proteins or, alternatively, small molecules that specifically alter protein stability or trafficking. Unfortunately, when studying these drug candidates, classical biochemical approaches are prone to artifacts. The apurinic/apyrimidinic endonuclease (APE1) protein is an essential component of the base excision repair (BER) pathway that is responsible for repairing DNA damage caused by oxidative and alkylating agents. In this work, we combined conditional gene expression knockdown of APE1 protein by RNA interference (RNAi) technology with re-expression of an ectopic recombinant form of APE1 fused with the photoconvertible fluorescent protein (PCFP) Dendra2. Dendra2 did not alter the subcellular localization or endonuclease activity of APE1. We calculated APE1 half-life and compared these results with the classical biochemical approach, which is based on cycloheximide (CHX) treatment. In conclusion, we combined RNAi and in vivo confocal microscopy to study a DNA repair protein demonstrating the feasibility and the advantage of this approach for the study of the cellular dynamic of a DNA repair protein.",
keywords = "APE1/Ref-1, Dendra2, Live confocal microscopy, Protein half-life, RNAi",
author = "Gianluca Tell and {Di Piazza}, Matteo and Malgorzata Kamocka and Carlo Vascotto",
year = "2013",
month = "10",
doi = "10.2144/000114088",
language = "English",
volume = "55",
pages = "198--203",
journal = "BioTechniques",
issn = "0736-6205",
publisher = "Eaton Publishing Company",
number = "4",

}

TY - JOUR

T1 - Combining RNAi and in vivo confocal microscopy analysis of the photoconvertible fluorescent protein Dendra2 to study a DNA repair protein

AU - Tell, Gianluca

AU - Di Piazza, Matteo

AU - Kamocka, Malgorzata

AU - Vascotto, Carlo

PY - 2013/10

Y1 - 2013/10

N2 - Clinical approaches for tumor treatment often rely on combination therapy where a DNA damaging agent is used in combination with a DNA repair protein inhibitor. For this reason, great efforts have been made during the last decade to identify inhibitors of DNA repair proteins or, alternatively, small molecules that specifically alter protein stability or trafficking. Unfortunately, when studying these drug candidates, classical biochemical approaches are prone to artifacts. The apurinic/apyrimidinic endonuclease (APE1) protein is an essential component of the base excision repair (BER) pathway that is responsible for repairing DNA damage caused by oxidative and alkylating agents. In this work, we combined conditional gene expression knockdown of APE1 protein by RNA interference (RNAi) technology with re-expression of an ectopic recombinant form of APE1 fused with the photoconvertible fluorescent protein (PCFP) Dendra2. Dendra2 did not alter the subcellular localization or endonuclease activity of APE1. We calculated APE1 half-life and compared these results with the classical biochemical approach, which is based on cycloheximide (CHX) treatment. In conclusion, we combined RNAi and in vivo confocal microscopy to study a DNA repair protein demonstrating the feasibility and the advantage of this approach for the study of the cellular dynamic of a DNA repair protein.

AB - Clinical approaches for tumor treatment often rely on combination therapy where a DNA damaging agent is used in combination with a DNA repair protein inhibitor. For this reason, great efforts have been made during the last decade to identify inhibitors of DNA repair proteins or, alternatively, small molecules that specifically alter protein stability or trafficking. Unfortunately, when studying these drug candidates, classical biochemical approaches are prone to artifacts. The apurinic/apyrimidinic endonuclease (APE1) protein is an essential component of the base excision repair (BER) pathway that is responsible for repairing DNA damage caused by oxidative and alkylating agents. In this work, we combined conditional gene expression knockdown of APE1 protein by RNA interference (RNAi) technology with re-expression of an ectopic recombinant form of APE1 fused with the photoconvertible fluorescent protein (PCFP) Dendra2. Dendra2 did not alter the subcellular localization or endonuclease activity of APE1. We calculated APE1 half-life and compared these results with the classical biochemical approach, which is based on cycloheximide (CHX) treatment. In conclusion, we combined RNAi and in vivo confocal microscopy to study a DNA repair protein demonstrating the feasibility and the advantage of this approach for the study of the cellular dynamic of a DNA repair protein.

KW - APE1/Ref-1

KW - Dendra2

KW - Live confocal microscopy

KW - Protein half-life

KW - RNAi

UR - http://www.scopus.com/inward/record.url?scp=84885440582&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84885440582&partnerID=8YFLogxK

U2 - 10.2144/000114088

DO - 10.2144/000114088

M3 - Article

C2 - 24107251

AN - SCOPUS:84885440582

VL - 55

SP - 198

EP - 203

JO - BioTechniques

JF - BioTechniques

SN - 0736-6205

IS - 4

ER -