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An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping
Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854293/ https://www.ncbi.nlm.nih.gov/pubmed/29543903 http://dx.doi.org/10.1371/journal.pone.0193180 |
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author | Lambert, Christopher J. Freshner, Briana C. Chung, Arlen Stevenson, Tamara J. Bowles, D. Miranda Samuel, Raheel Gale, Bruce K. Bonkowsky, Joshua L. |
author_facet | Lambert, Christopher J. Freshner, Briana C. Chung, Arlen Stevenson, Tamara J. Bowles, D. Miranda Samuel, Raheel Gale, Bruce K. Bonkowsky, Joshua L. |
author_sort | Lambert, Christopher J. |
collection | PubMed |
description | Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype. Our objective was to develop an automated system that would rapidly obtain cells and DNA from zebrafish embryos and larvae for genotyping, and that would keep the animals alive. We describe the development, testing, and validation of a zebrafish embryonic genotyping device, termed “ZEG” (Zebrafish Embryo Genotyper). Using microfluidic harmonic oscillation of the animal on a roughened glass surface, the ZEG is able to obtain genetic material (cells and DNA) for use in genotyping, from 24 embryos or larvae simultaneously in less than 10 minutes. Loading and unloading of the ZEG is performed manually with a standard pipette tip or transfer pipette. The obtained genetic material is amplified by PCR and can be used for subsequent analysis including sequencing, gel electrophoresis, or high-resolution melt-analysis. Sensitivity of genotyping and survival of animals are both greater than 90%. There are no apparent effects on body morphology, development, or motor behavior tests. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising. |
format | Online Article Text |
id | pubmed-5854293 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-58542932018-03-28 An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping Lambert, Christopher J. Freshner, Briana C. Chung, Arlen Stevenson, Tamara J. Bowles, D. Miranda Samuel, Raheel Gale, Bruce K. Bonkowsky, Joshua L. PLoS One Research Article Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype. Our objective was to develop an automated system that would rapidly obtain cells and DNA from zebrafish embryos and larvae for genotyping, and that would keep the animals alive. We describe the development, testing, and validation of a zebrafish embryonic genotyping device, termed “ZEG” (Zebrafish Embryo Genotyper). Using microfluidic harmonic oscillation of the animal on a roughened glass surface, the ZEG is able to obtain genetic material (cells and DNA) for use in genotyping, from 24 embryos or larvae simultaneously in less than 10 minutes. Loading and unloading of the ZEG is performed manually with a standard pipette tip or transfer pipette. The obtained genetic material is amplified by PCR and can be used for subsequent analysis including sequencing, gel electrophoresis, or high-resolution melt-analysis. Sensitivity of genotyping and survival of animals are both greater than 90%. There are no apparent effects on body morphology, development, or motor behavior tests. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising. Public Library of Science 2018-03-15 /pmc/articles/PMC5854293/ /pubmed/29543903 http://dx.doi.org/10.1371/journal.pone.0193180 Text en © 2018 Lambert et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Lambert, Christopher J. Freshner, Briana C. Chung, Arlen Stevenson, Tamara J. Bowles, D. Miranda Samuel, Raheel Gale, Bruce K. Bonkowsky, Joshua L. An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping |
title | An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping |
title_full | An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping |
title_fullStr | An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping |
title_full_unstemmed | An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping |
title_short | An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping |
title_sort | automated system for rapid cellular extraction from live zebrafish embryos and larvae: development and application to genotyping |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854293/ https://www.ncbi.nlm.nih.gov/pubmed/29543903 http://dx.doi.org/10.1371/journal.pone.0193180 |
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