title: TALEN-mediated gene editing of the thrombospondin-1 locus in axolotl.
abstract: 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.
authors: Brigham Regenerative Medicine Center and Department of Orthopedic Surgery Brigham and Women's Hospital Harvard Medical School Harvard Stem Cell Institute Cambridge Massachusetts 02139 USA.
authors: Tzu-Hsing
authors: TH
authors: Kuo
authors: Department of Genetics Development and Cell Biology Iowa State University Ames Iowa 50011 USA.
authors: Johanna E
authors: JE
authors: Kowalko
authors: Brigham Regenerative Medicine Center and Department of Orthopedic Surgery Brigham and Women's Hospital Harvard Medical School Harvard Stem Cell Institute Cambridge Massachusetts 02139 USA.
authors: Tia
authors: T
authors: DiTommaso
authors: Brigham Regenerative Medicine Center and Department of Orthopedic Surgery Brigham and Women's Hospital Harvard Medical School Harvard Stem Cell Institute Cambridge Massachusetts 02139 USA.
authors: Mandi
authors: M
authors: Nyambi
authors: Brigham Regenerative Medicine Center and Department of Orthopedic Surgery Brigham and Women's Hospital Harvard Medical School Harvard Stem Cell Institute Cambridge Massachusetts 02139 USA.
authors: Daniel T
authors: DT
authors: Montoro
authors: Department of Genetics Development and Cell Biology Iowa State University Ames Iowa 50011 USA.
authors: Jeffrey J
authors: JJ
authors: Essner
authors: Brigham Regenerative Medicine Center and Department of Orthopedic Surgery Brigham and Women's Hospital Harvard Medical School Harvard Stem Cell Institute Cambridge Massachusetts 02139 USA.
authors: Jessica L
authors: JL
authors: Whited
keywords: Axolotl; TALEN; gene targeting; genome editing; limb; regeneration; thrombospondin‐1