A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Journal of Visualized Experiments, 2011 · DOI: 10.3791/2713 · Published: June 22, 2011

Simple Explanation

Newts can regenerate lost body parts, including the lens of the eye. When the lens is removed, cells from the dorsal iris transdifferentiate into lens cells, forming a new lens in about a month. The regeneration potential of iris cells can be studied by transplanting cultured iris pigment epithelial (IPE) cells. Dorsal iris cells maintain their lens-forming capacity, while ventral iris cells do not. This system of determining regeneration potential is useful for studying the genes and proteins involved in lens regeneration.

Study Duration

2 weeks

Participants

7 newts

Evidence Level

Not specified

Key Findings

1
Dorsal iris reaggregates maintain lens-forming capacity, while ventral aggregates do not, mimicking the in vivo process.
2
Overexpression of six-3 in ventral iris cells, along with retinoic acid treatment, can induce lens formation.
3
Cultured iris cells can be transfected with genes to examine their function in lens regeneration.

Research Summary

This protocol establishes an in vitro system to study lens regeneration mechanisms in newts, allowing for gain-of-function and loss-of-function experiments. The aggregates, either dorsal or ventral, faithfully follow their in vivo behavior during regeneration. The aggregates or the irises as a whole can easily be treated with growth factors and examine their effects as described in this protocol.

Practical Implications

Study Gene Function

This technique allows for the study of specific genes that contribute towards the lens regeneration mechanism in newt eye.

Drug Discovery

The system enables the testing of growth factors and other molecules to examine their effects on lens regeneration.

Reduce Animal Use

This in vitro system can alleviate the tremendous effort required for transgenesis in newts.

Study Limitations

1
The study focuses solely on newts, limiting generalizability to other species.
2
The in vitro system may not fully replicate the complex in vivo environment.
3
Long-term effects of gene transfection and growth factor treatment were not assessed.

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