The zebrafish is an increasingly popular laboratory organism with many experimental advantages. The animal model has been used by biologists to discover the detailed processes that underlie vertebrate development and disease, and study vertebrate haematopoiesis and immune cell interactions. The zebrafish embryo is also a popular vertebrate model for
Zebrafish research, however, is limited by a paucity of good antibody reagents that are essential tools for determining the precise details of biological processes. 1 Gavin James Wright, Sanger Institute, Cambridge, United Kingdom (http://grantome.com/grant/NIH/R01-NS063400-01). Consequently, zebrafish researchers have historically been plagued by a paucity of robust antibodies in zebrafish.
In my view, the advent of CRISPR/Cas9-mediated gene editing in zebrafish makes it possible to circumvent this problem by utilizing gene editing technologies to introduce epitope tags and fluorescent markers at native loci to enable accurate and robust localization and isolation of genes of interest in zebrafish and other organisms (Hoshijima et al., 2016).
The edited ntlV5 allele is expressed faithfully (B-E). The expression pattern of the V5 antigen in (B) mid- and (D) late-gastrula ntlV5/+ embryos revealed by immunohistochemistry mimics the expression of ntl transcripts detected by in situ hybridization in comparably staged embryos (C, E). Image source: Hoshijima, K., Jurynec, M. J., & Grunwald, D. J. (2016). Precise editing of the zebrafish genome made simple and efficient. Developmental cell, 36(6), 654-667.
In this experiment, the V5 epitope tag is detectable by common commercially available antibodies. The use of a known epitope sequence that is precisely integrated into the genome streamlines validation of expression patterns in genes of interest, bypassing issues including proprietary epitope sequences and variability in antibody generation methods.
A knock-in GFP allele of ntl drives reporter expression in vivo (zebrafish embryo, 24 hpf). Image source: Hoshijima, K., Jurynec, M. J., Grunwald, D. J. (2016). Precise editing of the zebrafish genome made simple and efficient. Developmental cell, 36(6), 654-667.
In addition to addressing availability of robust antibodies, advances in gene editing technology will enable researchers to revisit long-standing issues of reproducibility in the biological sciences on a larger scale, potentially through revamping of the antibody validation pipeline. Utilizing CRISPR/Cas9 to generate knock-out and knock-in animal strains and cell lines for use in antibody validation experiments is one potential application in this arena. New and emerging variations on this gene editing technology will likely be a key player in ongoing discussions of reproducibility in the biological sciences.
- Wright, Gavin James; Sanger Institute, Cambridge, United Kingdom (http://grantome.com/grant/NIH/R01-NS063400-01)
- Dobson, J. T., Da’as, S., McBride, E. R., & Berman, J. N. (2009). Fluorescence‐activated cell sorting (FACS) of whole mount in situ hybridization (WISH) labelled haematopoietic cell populations in the zebrafish. British journal of haematology, 144(5), 732-735.
- Staudt, N., Müller-Sienerth, N., Fane-Dremucheva, A., Yusaf, S. P., Millrine, D., & Wright, G. J. (2015). A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining. Biochemical and biophysical research communications, 456(1), 527-533.
- Hoshijima, K., Jurynec, M. J., & Grunwald, D. J. (2016). Precise editing of the zebrafish genome made simple and efficient. Developmental cell, 36(6), 654-667.
About the Author: Ben Jussila
|x||Ben is an R&D technician at NemaMetrix, specializing in genome editing with CRISPR/Cas9 technology in zebrafish. He received his Bachelor of Science from the University of Minnesota in Genetics, Cell Biology & Development in 2014, and worked as a research technician at the University of Utah prior to joining the NemaMetrix team in 2018. He is currently completing his masters in biology online at the University of Nebraska, Kearney. Ben is passionate about nature and conservation, and in his spare time enjoys science outreach, field herpetology, his cats, and raising and breeding reptiles and amphibians.|