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TALEN-Mediated Gene Editing of the Thrombospondin-1 Locus in Axolotl
  • Tzu-Hsing Kuo, Harvard Medical School
  • Johanna E. Kowalko, Iowa State University
  • Tia DiTommaso, Harvard Medical School
  • Mandi Nyambi, Harvard Medical School
  • Daniel T. Montoro, Harvard Medical School
  • Jeffrey J. Essner, Iowa State University
  • Jessica L. Whited, Harvard Medical School
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Loss-of-function genetics provides strong evidence for a gene's function in a wild-type context. In many model systems, this approach has been invaluable for discovering the function of genes in diverse biological processes. Axolotls are urodele amphibians (salamanders) with astonishing regenerative abilities, capable of regenerating entire limbs, portions of the tail (including spinal cord), heart, and brain into adulthood. With their relatively short generation time among salamanders, they offer an outstanding opportunity to interrogate natural mechanisms for appendage and organ regeneration provided that the tools are developed to address these long-standing questions. Here we demonstrate targeted modification of the thrombospondin-1 (tsp-1) locus using transcription-activator-like effector nucleases (TALENs) and identify a role of tsp-1 in recruitment of myeloid cells during limb regeneration. We find that while tsp-1-edited mosaic animals still regenerate limbs, they exhibit a reduced subepidermal collagen layer in limbs and an increased number of myeloid cells within blastemas. This work presents a protocol for generating and genotyping mosaic axolotls with TALEN-mediated gene edits.

This article is from Regeneration 2 (2015): 37, doi:10.1002/reg2.29. Posted with permission.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Tzu-Hsing Kuo, et al
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Tzu-Hsing Kuo, Johanna E. Kowalko, Tia DiTommaso, Mandi Nyambi, et al.. "TALEN-Mediated Gene Editing of the Thrombospondin-1 Locus in Axolotl" Regeneration Vol. 2 Iss. 1 (2015) p. 37 - 43
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