Visualization of C. elegans transgenic arrays by GFP

Area of Science:

• Cell Biology
• Genetics
• Developmental Biology

Background:

• Green fluorescent protein (GFP) coupled with the E. coli lac repressor (LacI) targets specific DNA sequences (lacO) for chromosome visualization in various cell types.
• Genetic mosaic analysis requires cell-autonomous markers for single-cell resolution, a capability not fully realized in previous methods.
• The C. elegans lin-3 gene, an EGF family member, is crucial for vulval development, acting via LET-23 signaling, with potential roles in later vulval cell signaling.

Purpose of the Study:

• To establish a robust system for visualizing transgenes and chromosomes in living C. elegans.
• To validate the utility of the GFP-LacI*lacO system for high-resolution genetic mosaic analysis.
• To precisely determine the site of lin-3 action during vulval induction using mosaic analysis.

Main Methods:

• Engineered C. elegans to express a nuclear-localized GFP-LacI fusion protein.
• Incorporated a 256-copy lacO repeat sequence into transgenes for visualization.
• Performed genetic mosaic analysis using the lin-3 gene as a model to assess the system's efficacy.

Main Results:

• Successfully visualized transgenes and chromosomes in living C. elegans using the GFP-LacI*lacO system, appearing as nuclear-localized bright spots.
• Demonstrated the system's capability to detect polyploidy and analyze chromosome segregation.
• Confirmed lin-3 acts in the anchor cell to induce vulva development, validating the system's utility for transgene detection.

Conclusions:

• The GFP-LacI*lacO system is effective for visualizing chromosomes and extrachromosomal transgenes in C. elegans.
• This system serves as a valuable marker for high-resolution genetic mosaic analysis, enabling rapid assessment of transgene loss.
• Established that the lin-3 gene is essential in the anchor cell for inducing vulval precursor cell development.