FLP-mediated conditional loss of an essential gene to facilitate complementation assays Page: I
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Ganesan, Savita. FLP-mediated conditional loss of an essential gene to facilitate
complementation assays. Master of Science (Biochemistry), December 2007, 117 pp., 11 tables,
34 illustrations, bibliography, 53 titles.
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, the newly developed 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 (the first vector), a wild-type version of the gene is
placed between target sequences recognized by FLP recombinase from the yeast 2 m 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 (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. The 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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Ganesan, Savita. FLP-mediated conditional loss of an essential gene to facilitate complementation assays, thesis, December 2007; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc5180/m1/2/: accessed July 15, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .