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Generation and Molecular Characterization of Transient tet1/2/3 Zebrafish Knockouts

Abstract

5-methylcytosine (5mC) is a gene-regulatory mark associated with transcriptional repression. 5mC can be erased through the catalytic action of Ten-eleven translocation (TET) methylcytosine dioxygenases (TET1, TET2, TET3), which oxidize 5mC resulting in its removal from the genome. In vertebrates, TET enzymes facilitate DNA demethylation of regulatory regions linked to genes involved in developmental processes. Consequently, TET ablation leads to severe morphological defects and developmental arrest. Here we describe a system that can facilitate the study of relationships between TET enzymes, 5mC, and embryo development. We provide detailed descriptions for the generation of F0 zebrafish tet1/2/3 knockouts using CRISPR/Cas9 technology and elaborate on the strategies to assess the impact of TET loss by reduced representation bisulfite sequencing (RRBS).

Type Book section
ISBN 1940-6029 (Electronic) 1064-3745 (Linking)
Authors Ross, S. E.; Bogdanovic, O.
Responsible Garvan Author A/Prof Ozren Bogdanovic
Publisher Name Methods in Molecular Biology
Published Date 2021-05-31
Published Volume 2272
Published Pages 281-317
Status Published in-print
DOI 10.1007/978-1-0716-1294-1_17
URL link to publisher's version https://www.ncbi.nlm.nih.gov/pubmed/34009621