FLP-mediated conditional loss of an essential gene to facilitate complementation assays

Description:

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 μm 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.

Creator(s): Ganesan, Savita
Creation Date: December 2007
Partner(s):
UNT Libraries
Collection(s):
UNT Theses and Dissertations
Usage:
Total Uses: 271
Past 30 days: 17
Yesterday: 1
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Publisher Info:
Publisher Name: University of North Texas
Place of Publication: Denton, Texas
Date(s):
  • Creation: December 2007
  • Digitized: February 12, 2008
Description:

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 μm 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.

Degree:
Level: Master's
Discipline: Biochemistry
Language(s):
Subject(s):
Keyword(s): FLP/FRT recombination | complementation assay | ATSUC2
Contributor(s):
Partner:
UNT Libraries
Collection:
UNT Theses and Dissertations
Identifier:
  • OCLC: 228010560 |
  • ARK: ark:/67531/metadc5180
Resource Type: Thesis or Dissertation
Format: Text
Rights:
Access: Public
License: Copyright
Holder: Ganesan, Savita
Statement: Copyright is held by the author, unless otherwise noted. All rights reserved.