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Gene inactivation by multiphoton-targeted photochemistry
1Beckman Laser Institute and Medical Clinic and Center for Biomedical Engineering, University of California, Irvine 92612-1475, USA. mberns@bli.uci.edu
Abstract:
Multiphoton-targeted photochemistry was used to selectively inactivate the expression of genes in vertebrate cells. A membrane permeable DNA-associating vital dye, ethidium bromide monoacetate (visible wavelength single photon absorption peak at 530 nm) was used to photosensitize chromosomes in dividing cells. A 100-ps infrared laser beam operating at 1.06 microns was focused onto a selected region of a mitotic chromosome corresponding to the sites of the nucleolar (ribosomal) genes. Individual cells followed through mitosis demonstrated a reduction in the number of nucleoli formed in daughter cells that corresponded to the number of nucleolar genes sites irradiated. These results demonstrate the ability to selectively manipulate genes by using the focal point specificity characteristic of multiphoton microscopy. This technique should have wide biotechnology applications both in vitro and in vivo.
Insights
Multiphoton-targeted photochemistry precisely inactivates gene expression in vertebrate cells. This method uses a laser and photosensitizing dye to target specific gene sites on chromosomes, impacting cell development.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Gene expression regulation is crucial for cellular function and development.
- Targeted gene manipulation is essential for understanding gene function and disease.
- Existing methods for gene inactivation may lack precision or specificity.
Purpose of the Study:
- To develop and demonstrate a method for selective gene inactivation in vertebrate cells.
- To utilize multiphoton microscopy for precise targeting of specific gene loci.
- To assess the efficacy of targeted photochemistry in reducing gene expression.
Main Methods:
- Employing multiphoton-targeted photochemistry with a photosensitizing dye (ethidium bromide monoacetate).
- Using a pulsed infrared laser to focus on specific regions of mitotic chromosomes, targeting nucleolar (ribosomal) genes.
- Tracking individual cells through mitosis to observe the effects of gene site irradiation on daughter cells.
Main Results:
- Selective reduction in nucleoli formation in daughter cells, correlating with the number of targeted nucleolar gene sites.
- Demonstrated precise gene inactivation at the chromosomal level using focal point specificity.
- Validated the ability to manipulate gene expression through targeted photochemistry.
Conclusions:
- Multiphoton-targeted photochemistry offers a highly specific method for gene inactivation in vertebrate cells.
- The technique leverages the precision of multiphoton microscopy for targeted genetic manipulation.
- This approach holds significant potential for biotechnology applications, both in vitro and in vivo.