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https://www.selleckchem.com/products/enfortumab-vedotin-ejfv.html 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).TET family enzymes normally oxidize 5-methylcytosine (5mC) in DNA, and play critical roles in shaping the epigenome. Despite their importance, assessing TET activity can be difficult, particularly given the challenge of studying modifications to single nucleobases within complex DNA substrates. We recently demonstrated that in addition to acting on 5mC, TET enzymes can act promiscuously on unnatural nucleobases. Here, we describe how these alternative unnatural substrates can be employed in facile assays to detect and quantify TET activity. DNA containing unnatural 5-vinylcytosine (vC) can be used as a direct endpoint reporter of TET activity, a method that can potentially be adapted to high-throughput platforms. Complementarily, DNA containing unnatural 5-ethynylcytosine (eyC) can trap and inactivate TET enzymes upon reaction, a strategy that can be used to extract active TET enzymes from a complex cellular milieu. We present a detailed PCR-based protocol to synthesize DNA probes with either natural or unnatural modifications, and methods for using these
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