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Use of PCR template-derived probes prevents off-target whole mount in situ hybridization in transgenic zebrafish.

Authors
Type
Published Article
Journal
Zebrafish
Publication Date
Volume
9
Issue
2
Pages
85–89
Identifiers
DOI: 10.1089/zeb.2011.0731
PMID: 22715949
Source
Medline

Abstract

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.

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