Quantitative evaluation of morpholino‐mediated protein knockdown of GFP, MSX1, and PAX7 during tail regeneration in Ambystoma mexicanum

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Abstract
Vertebrate regeneration is a fascinating but poorly understood biological phenomena. Urodele amphibians such as Ambystoma mexicanum (the axolotl) can functionally regenerate complex body structures such as the limb and tail, including the spinal cord, throughout life. So far, molecular studies on regeneration have been limited due to the paucity of tools for knocking-down gene and protein function. In this article, we quantitatively assessed the ability of morpholinos to specifically down-regulate protein expression in both cultured urodele cells and in vivo. We focused on the down-regulation of green fluorescent protein and two axolotl proteins, MSX1 and PAX7. Our data show that the expression of these proteins can be efficiently reduced by morpholinos. MSX1 has been hypothesized to be involved in muscle dedifferentiation based on experiments using cultured myotubes. Our studies in in vivo muscle fibers so far have shown no influence of overexpressing or down-regulating MSX1 on the dedifferentiation process. Developmental Dynamics 232:162–170, 2005.