Computational protocol: Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish

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Protocol publication

[…] CRISPR col12a1a and col12a1b gRNA were designed using a combination of different webtools: ZiFit (http://zifit.partners.org/ZiFiT), CRISPR Design (http://crispr.mit.edu), and Mojo Hand (http://talendesign.org). gRNA expression vectors were built by ligation of annealed oligonucleotides into pT7-gRNA expression vector (Addgene #46759) as described previously. Capped sense nls-zCas9-nls RNA was synthesized using mMESSAGE mMACHINE T3 kit (Ambion) and pT3TS-nCas9n (Addgene # 46757) as template. RNA was purified using RNAeasy Mini Kit (Qiagen). gRNA was in vitro transcribed from gRNA expression vectors using mMESSAGE mMACHINE T7 kit and purified using mirVana miRNA isolation kit (Ambion). All synthesized RNAs were assessed for size and quality by gel electrophoresis. A mix of 75 pg gRNA and 150 pg nls-zCas9-nls RNA was microinjected into one-cell-stage embryos to determine efficiency of individual gRNAs to introduce mutations in the target site in pools of 24 hpf embryos using restriction fragment length polymorphisms (RFLP) analysis. For subsequent experiments we used a combination (75 pg each) of col12a1a gRNA#1 (target sequence 5′-GGCTGTGGTTCAGTACAGCT-3′; targeting exon 6) and col12a1b gRNA#1 (target sequence 5′- GGTCAGGCATTTGGCAGCGG-3′; targeting exon 2) and 150 pg nls-zCas9-nls RNA for microinjection. A previously described gRNA targeting GFP (target sequence: 5′GGCGAGGGCGATGCCACCTA-3) served as control. Efficient mutagenesis of target loci was confirmed after each injection by RFLP analysis as follows. For col12a1a gRNA#1, a 308 bp fragment was amplified using primers 5′- TGGAGTGTGGGAAGAGAAAACTT-3′ and 5′- CTAATGAGAATTTGTCGGCAGCG-3′ and digested with AluI. For col12a1b gRNA#1, a 400 bp fragment was amplified using primers 5′-TGGAGCATGTATTTTCCCCTTGA-3′ and 5′-GCTCCAGTCCTTTTGTTCATTCC-3′ and digested with AciI. [...] For live confocal imaging, zebrafish larvae were anesthetized in PBS containing 0.02% MS222 and mounted in the appropriate orientation in 1%low melting point agarose (UltraPureTM, Invitrogen). During imaging the larvae were covered with 0.01% MS222-containing fish water to keep preparations from drying out. For time-lapse imaging, agarose covering the lesion site was gently removed after gelation. Time-lapse imaging was performed for 19 h starting at 6 hpl. After initial visual inspection, acquired time-lapse images were denoised to improve image quality (enhance detail). Background noise, caused by the low signal to noise ratio, was removed from acquired time-lapse images using the ImageJ plugin CANDLE-J algorithm, which allows to remove the noise compartment while preserving the structural information with high fidelity. We verified that this procedure did not remove signals from processes of low fluorescence intensity by comparing raw movies with CANDLE-J-processed movies, which showed that that edges of features remained conserved after denoising. […]

Pipeline specifications

Software tools ImageJ, CANDLE
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Danio rerio
Diseases Spinal Injuries