Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration.
The analysis of newt lens regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of lens regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into lens cells in newt lens regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene-transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in lens regeneration, Pax6, which is suggested to be involved in normal eye development and lens regeneration, was transfected. Ectopic expression of lens-specific crystallins was obtained in cells that show no such activity in normal lens regeneration. These results made it possible for the first time to analyze the molecular mechanism of lens regeneration in the adult newt.
Hayashi
T
T
Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
Yamagishi
A
A
Kuroiwa
A
A
Mizuno
N
N
Kondoh
H
H
Okamoto
M
M
D000818:Animals; D002411:Cation Exchange Resins / Q000378:metabolism; D017690:Cell Transplantation; D002478:Cells, Cultured; D003459:Crystallins / Q000235:genetics / Q000378:metabolism; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D007202:Indicators and Reagents / Q000378:metabolism; D007498:Iris / Q000166:cytology; D007908:Lens, Crystalline / Q000033:anatomy & histology / Q000502:physiology*; D050356:Lipid Metabolism; D008055:Lipids; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D010857:Pigment Epithelium of Eye / Q000166:cytology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012038:Regeneration / Q000235:genetics* / Q000502:physiology; D012097:Repressor Proteins; D012455:Salamandridae / Q000502:physiology*; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D002411:Cation Exchange Resins; D003459:Crystallins; D005136:Eye Proteins; D018398:Homeodomain Proteins; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors; D012097:Repressor Proteins; D001616:beta-Galactosidase