Large Scale Technologies and Resources
Ganesan, Savita , Ayre, Brian G. .
FLP-mediated Conditional Loss of an Essential Gene to Facilitate Complementation Assays.
We have established a two-component vector system to facilitate high-throughput complementation assays of essential genes by generating on-demand null mutant plants. Commonly, when it is desirable to replace an essential gene with an allelic series of mutated genes, or genes with altered expression patterns, the complementing constructs are introduced into heterozygous plants, followed by the selection of homozygous null segregants. To overcome this laborious and time-consuming step, our two-component system utilizes a site-specific recombinase to excise a wild-type copy of the gene of interest from transformed tissues. In the first component (i.e., the first vector), a wild-type version of the gene is placed between target sequences recognized by FLP recombinase from the yeast 2 Āµm plasmid. This construct is transformed into a plant heterozygous for a null mutation at the endogenous locus, and progeny plants carrying the excisable complementing gene and segregating homozygous knockout at the endogenous locus are selected. The second component (i.e., the second vector) carries the experimental gene along with the FLP gene. When this construct is introduced, FLP recombinase excises the complementing gene, leaving the experimental gene as the only functional copy. TheĀ FLP gene is driven by an Egg Apparatus Specific Enhancer (EASE) to ensure excision of the complementing cDNA in the egg cell and zygote following floral-dip transformation. The utility of this system is being tested using various experimental derivatives of the essential sucrose-proton symporter, AtSUC2, which is required for photoassimilate transport.
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1 - University of North Texas, Biological Sciences, P O Box 305220, Denton, TX, 76203, USA
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM