crispr/cas9-mediated zebrafish knock-in as a novel strategy to study midbrain-hindbrain boundary development

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ID: 243427
2017
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Abstract
The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a. The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development.
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kesavan2017frontierscrispr/cas9-mediated Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors ;Gokul Kesavan;Avinash Chekuru;Anja Machate;Michael Brand
Journal Journal of medical systems
Year 2017
DOI
10.3389/fnana.2017.00052
URL
http://journal.frontiersin.org/article/10.3389/fnana.2017.00052/full
Keywords
crispr/cas9 biological psychiatry neuropsychiatryhuman anatomy zebrafish and transgenesisneurosciences

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