TALEN‐mediated gene editing of the thrombospondin‐1 locus in axolotl

Regeneration

Tzu-Hsing Kuo¹  Johanna E. Kowalko²  Tia DiTommaso¹  Mandi Nyambi¹  Daniel T. Montoro¹  Jeffrey J. Essner² 

[1] Brigham Regenerative Medicine Center and Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Harvard Stem Cell Institute, Cambridge, Massachusetts, USA;Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa, USA

关键词: Axolotl;    gene targeting;    genome editing;    limb;    regeneration;    TALEN;    thrombospondin‐1;   

DOI  :  10.1002/reg2.29

来源: Wiley

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 The Authors. Regeneration published by John Wiley & Sons Ltd.

Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.