ZFIN ID: ZDB-PUB-120702-35
Use of PCR template-derived probes prevents off-target whole mount in situ hybridization in transgenic zebrafish
Cha, Y.R., and Weinstein, B.M.
Date: 2012
Source: Zebrafish   9(2): 85-89 (Journal)
Registered Authors: Cha, Young, Weinstein, Brant M.
Keywords: none
MeSH Terms:
  • Animals
  • DNA Probes*
  • Embryo, Nonmammalian
  • In Situ Hybridization/methods
  • In Situ Hybridization/veterinary*
  • Polymerase Chain Reaction
  • Zebrafish/genetics*
PubMed: 22715949 Full text @ Zebrafish

Transgenic zebrafish have been utilized for in vivo analysis of cell behaviors using advanced imaging techniques, for analyzing spatiotemporal gene regulation, and for targeted mis-expression of transgenes. The Tg(fli1a:EGFP)y1 vascular reporter has been particularly useful for examining the development of blood and lymphatic vessels, but it has been suggested that whole-mount in situ hybridization may result high background staining in this line, potentially limiting its usefulness. Here, we show that off-target hybridization of plasmid vector-derived probes to tissues expressing transgenes occurs in a number of different commonly used transgenic lines as a result of multiple cloning site sequences present in the cloning vectors, suggesting this may be a more general problem. However, we also show that this problem is easily avoided by performing in situ hybridization using probes synthesized from PCR templates lacking vector sequences.