Analysis of LINE-1 retrotransposition at the single nucleus level

Pasano Bojang, Kenneth Ramos

Research output: Contribution to journalArticle

Abstract

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Original languageEnglish (US)
Article numbere53753
JournalJournal of Visualized Experiments
Volume2016
Issue number110
DOIs
StatePublished - Apr 1 2016

Keywords

  • Biochemistry
  • Chromosome spreads
  • FISH
  • HepG2
  • Issue 110
  • Line-1
  • Neomycin
  • Retrotransposon
  • Ribonucleoprotein particles (RNP)

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

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