Development of transgenic knockdown methods in Ambystoma mexicanum to study satellite cell activation during limb regeneration
Cell Biology
Final Report Abstract
In this project we developed CRISPR-mediated gene modification and gene knock-in in the regenerative salamander, Ambystoma mexicanum. We were able to demonstrate efficient modification of target genes by injection of Cas9-gRNA protein-RNA complexes into the axolotl egg. Generation of loss-of-function Pax7 alleles showed that the Pax7 gene is essential for limb muscle in the axolotl. In addition, we could insert long sequences of DNA by non-homologous end joining into targeted loci, which allowed us to knock-in gene sequences for tamoxifen inducible Cre-ERT recombinases into the Pax7 locus. Using this transgenic animal crossed to a loxP reporter strain, we were able to show that Pax7 satellite cells are the major source cells for muscle regeneration during limb regeneration.
Publications
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(2014). CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration. Stem Cell Reports, 3:444-459
Fei, Ji-Feng; Schuez, Maritta; Tazaki, Akira; Taniguchi, Yuka; Roensch, Kathleen & Tanaka, Elly M.
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(2014). Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination. Nat Protoc. 9:529-540
Khattak, Shahryar; Murawala, Prayag; Andreas, Heino; Kappert, Verena; Schuez, Maritta; Sandoval-Guzmán, Tatiana; Crawford, Karen & Tanaka, Elly M.
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(2016) Tissue and time-directed electroporation of CAS9 protein-gRNA complexes in vivo yields efficient multigene knockout for studying gene function in regeneration. Nature Regenerative Medicine, Jun 9;1:16002
Fei, Ji-Feng; Knapp, Dunja; Schuez, Maritta; Murawala, Prayag; Zou, Yan; Pal Singh, Sumeet; Drechsel, David & Tanaka, Elly M.
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(2017) Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration. PNAS, 114:12501-12506
Fei, Ji-Feng; Schuez, Maritta; Knapp, Dunja; Taniguchi, Yuka; Drechsel, David N. & Tanaka, Elly M.
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(2017). The axolotl genome and the evolution of key tissue formation regulators. Nature, 554(7690):50-55
Nowoshilow, Sergej; Schloissnig, Siegfried; Fei, Ji-Feng; Dahl, Andreas; Pang, Andy W. C.; Pippel, Martin; Winkler, Sylke; Hastie, Alex R.; Young, George; Roscito, Juliana G.; Falcon, Francisco; Knapp, Dunja; Powell, Sean; Cruz, Alfredo; Cao, Han; Habermann, Bianca; Hiller, Michael; Tanaka, Elly M. & Myers, Eugene W.
