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  4. Germline Transgenic Methods for Tracking Cells and Testing Gene Function during Regeneration in the Axolotl

Germline Transgenic Methods for Tracking Cells and Testing Gene Function during Regeneration in the Axolotl

Stem Cell Reports, 2013 · DOI: 10.1016/j.stemcr.2013.03.002 · Published: June 4, 2013

Regenerative MedicineGenetics

Simple Explanation

The salamander is the only tetrapod that regenerates complex body structures throughout life. This study introduces a comprehensive set of germline transgenic strains in the axolotl to facilitate cellular and molecular genetic dissection of regeneration. The researchers created transgenic axolotl lines with tissue-specific gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage. This allows for controlled study of how these tissues contribute to regeneration. The study also demonstrates the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types and inducibly overexpress the cell-cycle inhibitor p16INK4a, which negatively regulates spinal cord regeneration. These tools make the axolotl amenable for molecular analysis of regeneration.

Study Duration
Not specified
Participants
Laboratory-bred salamander Ambystoma mexicanum (axolotl)
Evidence Level
Resource

Key Findings

  • 1
    The study successfully established germline transgenic axolotl strains showing faithful, cell-type-specific expression of EGFP gene using tissue-specific promoters from different animal species.
  • 2
    The researchers demonstrated that muscle progenitors from a hand blastema can form upper- and lower-arm muscle by using CarAct:EGFP transgenic animal for revealing the potency of muscle to regenerate cells in all limb segments.
  • 3
    The study found that ERT2-Cre-ERT2 fusion provides tight, temporal control of gene expression in the axolotl and demonstrated cell-type and temporal control of gene expression is efficient and possible in germline transgenic axolotl lines.

Research Summary

This study introduces a comprehensive set of germline transgenic strains in the laboratory-bred salamander Ambystoma mexicanum (axolotl) that open up the cellular and molecular genetic dissection of regeneration. The researchers demonstrate tissue-dependent control of gene expression in nerve, Schwann cells, oligodendrocytes, muscle, epidermis, and cartilage and the use of tamoxifen-induced Cre/loxP-mediated recombination to indelibly mark different cell types. The study shows inducible overexpression of the cell-cycle inhibitor p16INK4a negatively regulates spinal cord regeneration, making the axolotl amenable for molecular analysis of regeneration.

Practical Implications

Cell Tracking during Regeneration

The generated transgenic lines enable the tracking of specific cell types during axolotl regeneration, providing insights into their roles and behaviors.

Controlled Gene Expression

The Cre/loxP system allows for precise temporal and spatial control of gene expression, facilitating the study of gene function during regeneration and development.

Molecular Analysis of Regeneration

The developed tools and insights will help investigate the mechanistic basis of regeneration in a vertebrate and its restriction in other animals.

Study Limitations

  • 1
    Potential for leakiness of the inducer in the Cre/loxP system, requiring careful monitoring and control.
  • 2
    Position effects due to random transgene integration, necessitating thorough screening of F1 progeny for specific and complete expression.
  • 3
    Quality of local water supply impacting ERT2-Cre-ERT2 system performance.

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