Gene Traps: A Powerful tool for Identification of Unknown Genes.

gene traps:

http://www.witrans.uni-frankfurt.de/forschungsbericht/f21/i82/p341/P2439.htm

enhancer trap:
http://www.arabidopsis.org/cshl-course/10-enhancer.html

gene trap:
http://www.arabidopsis.org/cshl-course/10-enhancer.html

The Arabidopsis research community has invested substantially in using insertional mutation (knockout library) and expression patterns (microarray experiments) to identify unknown genes. Unfortunately, the function of all unknown genes cannot be determined by these approaches alone for several reasons. First, disruption of functionally redundant genes may be without a phenotype if another gene somewhere else in the genome replaces it lost function. Second, many genes function at multiple stages of growth or may control more than one function. Disruption of such genes may be lethal or conceal their specific function in a single pathway. Expression analysis is ultimately limited by the source of tissue used to isolate the RNA probe. Transient or spatially limited expression are situations where genes are unlikely to be identified solely by analysis of expression patterns.

In a review entitled, “Gene Traps: Tools for Plant Development and Genomics”, published in the July, 2000 issue of Plant Cell, Dr. Patricia V. Springer of University of California at Riverside, outlines an alternative approach for identifying unknown gene called gene trapping which utilizes the random insertion of reporter gene constructs capable of responding to transcriptional signals to elucidate unknown gene function. Supported by illustrations, the author describes three basic types of gene traps: 1) enhancer trap (a promoterless reporter gene is expressed when an appropriate host promoter or enhancer activates it}, 2) promoter trap (when a reporter gene is inserted in an exon rendering the promoterless reporter gene transcriptionally active) and 3) gene trap (when a reporter gene is inserted in an intron resulting in a transcriptional fusion product at the splicing site)

The two vehicles for inserting the gene trap in plant tissues are either via T-DNA or by transposable element. Transposable elements utilize two main tagging strategies: selection for excision and selection for transposition. The reporter genes most often used these days are GUS (beta-glucrondiase), which requires an expensive substrate, or more recently GFP (green fluorescent protein) which does not require a substrate and can be used on live tissue. Unknown gene function is suggested by examination of the tissue for the temporal and spatial expression pattern of the reporter gene.

Besides gene identification, gene traps can also be used to identify promoters driving specific expression. The author gives an example as to how a root cap-specific promoter in an enhancer trap line was found to drive expression of a diphtheria toxin gene (DT-A) traps. Using the newly found promoter, the function of a particular gene can be expressed ectopically i.e., changing the organ specific expression of a gene by fusing the promoter with a structural gene of choice.

The author concludes that following the completion of sequencing of Arabidopsis genome, gene traps, are likely to play an increasingly important role in the next phase of Arabidopsis genomics – determining gene function.

 

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