Development of transgenic knockdown methods in Ambystoma mexicanum to study satellite cell activation during limb regeneration

• Applicant: Professorin Elly Margaret Tanaka, Ph.D.
• Subject Area: Developmental Biology ; Cell Biology
• Term: from 2007 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 54248322

• Final Report Year: 2020

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

• (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 JF, Schuez M, Tazaki A, Taniguchi Y, Roensch K, Tanaka EM
  
• (2014). Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination. Nat Protoc. 9:529-540
  Khattak, S., P. Murawala, H. Andreas, V. Kappert, M. Schuez, T. Sandoval-Guzman, K. Crawford, and E.M. Tanaka
  
• (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 JF, Knapp D, Schuez M, Murawala P, Zou Y, Singh P, Drechsel D, Tanaka EM
  
• (2017) Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration. PNAS, 114:12501-12506
  Fei JF, Schuez M, Knapp D, Taniguchi Y, Drechsel DN, Tanaka EM
  
• (2017). The axolotl genome and the evolution of key tissue formation regulators. Nature, 554(7690):50-55
  Nowoshilow S, Schloissnig S, Fei JF, Dahl A, Pang AWC, Pippe M, Winkler S, Hastie AR, Young G, Roscito JG, Falcon F, Knapp D, Powell S, Cruz A, Cao H, Habermann H, Hiller M, Tanaka EM, Myers EM