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A rapid method for temporally and spatially controlled CRISPR-mediated gene knockout in vertebrates will be an important tool to screen for genes involved in complex biological phenomena like regeneration. Here we show that in vivo injection of CAS9 protein-guide RNA (gRNA) complexes into the spinal cord lumen of the axolotl and subsequent electroporation leads to comprehensive knockout of Sox2 gene expression in SOX2+ neural stem cells with corresponding functional phenotypes from the gene knockout. This is particularly surprising considering the known prevalence of RNase activity in cerebral spinal fluid, which apparently the CAS9 protein protects against. The penetrance/efficiency of gene knockout in the protein-based system is far higher than corresponding electroporation of plasmid-based CRISPR systems. We further show that simultaneous delivery of CAS9-gRNA complexes directed against Sox2 and GFP yields efficient knockout of both genes in GFP-reporter animals. Finally, we show that this method can also be applied to other tissues such as skin and limb mesenchyme. This efficient delivery method opens up the possibility for rapid in vivo genetic screens during axolotl regeneration and can in principle be applied to other vertebrate tissue systems.
Pubmed ID: 29302334 RIS Download
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View all literature mentionsNon-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
View all literature mentionsTHIS RESOURCE IS NO LONGER IN SERVICE. Documented on May 5,2022.Tool that predicts interactions between transcription factors and their regulated genes from binding motifs. Understanding vertebrate development requires unraveling the cis-regulatory architecture of gene regulation. PRISM provides accurate genome-wide computational predictions of transcription factor binding sites for the human and mouse genomes, and integrates the predictions with GREAT to provide functional biological context. Together, accurate computational binding site prediction and GREAT produce for each transcription factor: 1. putative binding sites, 2. putative target genes, 3. putative biological roles of the transcription factor, and 4. putative cis-regulatory elements through which the factor regulates each target in each functional role.
View all literature mentionsAn antibody supplier which banks and distributes hybridomas and monoclonal antibodies for use in research. The bank includes antibodies against targets such as GFP, transcription factors, stem cells, and human.
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