PMID- 27167780
OWN - NLM
STAT- MEDLINE
DCOM- 20170707
LR  - 20181202
IS  - 1940-087X (Electronic)
IS  - 1940-087X (Linking)
IP  - 110
DP  - 2016 Apr 23
TI  - Analysis of LINE-1 Retrotransposition at the Single Nucleus Level.
LID - 10.3791/53753 [doi]
LID - 53753
AB  - 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.
FAU - Bojang, Pasano
AU  - Bojang P
AD  - Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, University of 
      Arizona College of Medicine; pbojang@email.arizona.edu.
FAU - Ramos, Kenneth S
AU  - Ramos KS
AD  - Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, University of 
      Arizona College of Medicine; Center for Applied Genetics and Genomic Medicine, 
      University of Arizona College of Medicine; ksramos@email.arizona.edu.
LA  - eng
GR  - P30 ES014443/ES/NIEHS NIH HHS/United States
GR  - R01 ES017274/ES/NIEHS NIH HHS/United States
PT  - Journal Article
PT  - Video-Audio Media
DEP - 20160423
PL  - United States
TA  - J Vis Exp
JT  - Journal of visualized experiments : JoVE
JID - 101313252
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Cell Nucleus/*genetics
MH  - DNA/genetics
MH  - Genome, Human
MH  - Hep G2 Cells
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/methods
MH  - *Long Interspersed Nucleotide Elements
PMC - PMC4941990
EDAT- 2016/05/12 06:00
MHDA- 2017/07/08 06:00
PMCR- 2018/04/23
CRDT- 2016/05/12 06:00
PHST- 2016/05/12 06:00 [entrez]
PHST- 2016/05/12 06:00 [pubmed]
PHST- 2017/07/08 06:00 [medline]
PHST- 2018/04/23 00:00 [pmc-release]
AID - 53753 [pii]
AID - 10.3791/53753 [doi]
PST - epublish
SO  - J Vis Exp. 2016 Apr 23;(110):53753. doi: 10.3791/53753.