PUBLICATION

Efficient discovery of ASCL1 regulatory sequences through transgene pooling

Authors
McGaughey, D.M., and McCallion, A.S.
ID
ZDB-PUB-100309-23
Date
2010
Source
Genomics   95(6): 363-369 (Journal)
Registered Authors
McCallion, Andy
Keywords
none
MeSH Terms
  • Animals
  • Basic Helix-Loop-Helix Transcription Factors/genetics*
  • Embryo, Nonmammalian
  • Gene Transfer Techniques
  • Humans
  • Regulatory Sequences, Nucleic Acid*
  • Transgenes
  • Zebrafish
  • Zebrafish Proteins/genetics*
PubMed
20206680 Full text @ Genomics
Abstract
Zebrafish transgenesis is a powerful and increasingly common strategy to assay vertebrate transcriptional regulatory control. Several challenges remain, however, to the broader application of this technique; they include increasing the rate with which transgenes can be analyzed and maximizing the informational value of the data generated. Presently, many rely on the injection of individual constructs and the analysis of resulting reporter expression in mosaic G0 embryos. Here, we contrast these approaches, examining whether injecting pooled transgene constructs can increase the efficiency with which regulatory sequences can be assayed, restricting analysis to the offspring of germ line transmitting transgenic zebrafish in an effort to reduce potential subjectivity. We selected a 64kb interval encompassing the human ASCL1 locus as our model interval and report the analysis of 9 highly conserved putative enhancers therein. We identified 32 transgene-positive zebrafish, transmitting one or more independent constructs displaying ASCL1-like regulatory control. Through examination of embryos harboring one or more transgenes, we demonstrate that five of the nine sequences account for the observed control and describe their likely roles in ASCL1 regulation. These data demonstrate the utility of this approach and its potential for further adaptation and higher throughput application.
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping