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

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.

SUBMITTER: Cha YR

PROVIDER: S-EPMC3388497 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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

Cha Young R YR Weinstein Brant M BM

Zebrafish 20120601 2

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 sh ...[more]

PMID: 22715949

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Project description:BackgroundDetection of unculturable bacteria and their localization in the host, by fluorescent in-situ hybridization (FISH), is a powerful technique in the study of host-bacteria interaction. FISH probes are designed to target the 16 s rRNA region of the bacteria to be detected. LNA probes have recently been used in FISH studies and proven to be more efficient. To date no report has employed LNA probes for FISH detection of bacterial endosymbiont in the whole mount tissues. Further, though speculated, bacteriocytes have not been reported from males of Bemisia tabaci.ResultsIn this study, we compared the efficiency in detecting bacteria by fluorescent DNA oligonucleotides versus modified probes containing Locked Nucleic Acid (LNA) substitution in their structure. We used the insect Bemisia tabaci as the experimental material since it carried simultaneous infection by two bacteria: one a primary endosymbiont, Portiera (and present in more numbers) while the other a secondary endosymbiont Arsenophonus (and present in less numbers). Thus a variation in the abundance of bacteria was expected. While detecting both the bacteria, we found a significant increase in the signal whenever LNA probes were used. However, the difference was more pronounced in detecting the secondary endosymbiont, wherein DNA probes gave weak signals when compared to LNA probes. Also, signal to noise ratio for LNA probes was higher than DNA probes. We found that LNA considerably improved sensitivity of FISH, as compared to the commonly used DNA oligonucleotide probe.ConclusionBy employing LNA probes we could detect endosymbiotic bacteria in males, which have never been reported previously. We were able to detect bacteriocytes containing Portiera and Arsenophonus in the males of B. tabaci. Thus, employing LNA probes at optimized conditions will help to significantly improve detection of bacteria at the lowest concentration and may give a comprehensible depiction about their specific distribution within samples.

Dataset Information

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

Publications

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

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