Visualization of C. elegans transgenic arrays by GFP

Abstract

Background: Targeting the green fluorescent protein (GFP) via the E. coli lac repressor (LacI) to a specific DNA sequence, the lac operator (lacO), allows visualization of chromosomes in yeast and mammalian cells. In principle this method of visualization could be used for genetic mosaic analysis, which requires cell-autonomous markers that can be scored easily and at single cell resolution. The C. elegans lin-3 gene encodes an epidermal growth factor family (EGF) growth factor. lin-3 is expressed in the gonadal anchor cell and acts through LET-23 (transmembrane protein tyrosine kinase and ortholog of EGF receptor) to signal the vulval precursor cells to generate vulval tissue. lin-3 is expressed in the vulval cells later, and recent evidence raises the possibility that lin-3 acts in the vulval cells as a relay signal during vulval induction. It is thus of interest to test the site of action of lin-3 by mosaic analysis. Results: We visualized transgenes in living C. elegans by targeting the green fluorescent protein (GFP) via the E. coli lac repressor (LacI) to a specific 256 sequence repeat of the lac operator (lacO) incorporated into transgenes. We engineered animals to express a nuclear-localized GFP-LacI fusion protein. C. elegans cells having a lacO transgene result in nuclear-localized bright spots (i.e., GFP-LacI bound to lacO). Cells with diffuse nuclear fluorescence correspond to unbound nuclear localized GFP-LacI. We detected chromosomes in living animals by chromosomally integrating the array of the lacO repeat sequence and visualizing the integrated transgene with GFP-LacI. This detection system can be applied to determine polyploidy as well as investigating chromosome segregation. To assess the GFP-LacI-lacO system as a marker for mosaic analysis, we conducted genetic mosaic analysis of the epidermal growth factor lin-3, expressed in the anchor cell. We establish that lin-3 acts in the anchor cell to induce vulva development, demonstrating this methods utility in detecting the presence of a transgene. Conclusions: The GFP-LacIlacO transgene detection system works in C. elegans for visualization of chromosomes and extrachromosomal transgenes. It can be used as a marker for genetic mosaic analysis. The lacO repeat sequence as an extrachromosomal array becomes a valuable technique allowing rapid, accurate determination of spontaneous loss of the array, thereby allowing high-resolution mosaic analysis. The lin-3 gene is required in the anchor cell to induce the epidermal vulval precursors cells to undergo vulval development.

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