Use of PCR template-derived probes prevents off-target whole mount in situ hybridization in transgenic 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.
Program in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institute of Health, Bethesda, Maryland 20892, USA.
Young R
YR
Cha
Brant M
BM
Weinstein
D000818:Animals; D015342:DNA Probes; D004625:Embryo, Nonmammalian; D017403:In Situ Hybridization / Q000379:methods / Q000662:veterinary*; D016133:Polymerase Chain Reaction; D015027:Zebrafish / Q000235:genetics*
D015342:DNA Probes