Exploring the role of four Kazald paralogs in Axolotl limb regeneration

Abstract

This study aimed to establish the foundation for understanding a broader inquiry into whether other Kazald paralogs function as compensatory mechanisms for the Kazald2 gene, recognized for its upregulated in blastema and potential critical role in the regeneration process (Bryant et al., 2017). Kazald2 morpholino morphants exhibited a noticeable delay in the regeneration process; however, researchers observed that knocking out the Kazald2 gene resulted in regeneration comparatively normal regeneration, suggesting the hypothesis of Gene Compensation among the Kazald paralogs. Gene compensation proposes that the other members of the Kazald paralogs may step in to fulfill the functions of Kazald2 when it is absent, thereby facilitating the regeneration process to proceed without significant impairment. The CRISPR-Cas9 system was employed to edit the genome of Kazald paralogs in axolotl embryos by inducing Double-Stranded Breaks. Gel electrophoresis analysis confirmed successful targeting of gene sequences, as evidenced by the presence of two distinct bands upon T7 digestion. This promising outcome suggests the feasibility of generating valuable Kazald mutants for Genetic Compensation research. However, it is important to acknowledge the potential challenge of mosaicism associated with CRISPR. Furthermore, while the Hybridization Chain Reaction experiment successfully executed the protocol, unfortunately, the newly designed probes did not yield the anticipated. This underscores the necessity for meticulous optimization of experimental factors such as probe concentration, hybridization conditions, and detection methods. Addressing these challenges is pivotal to enhancing the robustness and reliability of the experimental approach for future research applications.