Gene Traps: Tools for Plant Development and Genomics

Classical genetic approaches to gene identification rely on disruption of a gene leading to a recognizable phenotype. This approach continues to be an extremely successful one, yielding mutations that result in overt phenotypes reflecting the function of the corresponding gene. Not all genes, however, can be uncovered by mutagenesis for two main reasons. First, many genes are functionally redundant, sharing overlapping functions with other genes that may or may not be related at the sequence level. Mutation of a functionally redundant gene is not likely to lead to an easily recognizable phenotype, because one or more other family members can provide the same function. Analysis of systematic gene knockouts has revealed that a significant percentage of yeast genes have no obvious phenotype when disrupted, despite testing under a wide range of growth conditions (Ross-MacDonald et al., 1999). Therefore, it is likely that disruption of many plant genes will not result in an easily identifiable phenotype. Second, many genes function at multiple stages of development. Mutations in these genes may lead to early lethality or may be highly pleiotropic, which can mask the role of a gene in a specific pathway. A second common method of gene identification is based on expression patterns. In this case, genes that are expressed in spatially, temporally, or conditionally restricted patterns are isolated, traditionally via some type of differential screening approach. Recently, the differential mRNA display technique (Reuber and Ausubel, 1995) has increased the sensitivity of this approach. Additionally, recent developments with microarray and gene chip technologies allow the expression profiles of many genes to be analyzed (Richmond and Somerville, 2000). Nevertheless, these approaches are ultimately limited by the source of tissue used to isolate the RNA probe. Genes that are expressed transiently, at low levels, or in a small number of cells are unlikely to be identified. In recent years, techniques to identify genes have been developed that utilize random integration of reporter gene constructs. This approach has been called enhancer detection in Drosophila (reviewed in Bellen, 1999) and has also proven to be an extremely powerful tool in mouse developmental biology. In this review, I describe how enhancer detection systems have been adapted to plant biology so as to add to the arsenal of gene identification techniques available to the plant biology community. Several different types of “trapping” systems have been developed. The major difference among such systems lies in the reporter gene construct that is used. All these systems have been designed for gene identification, and the generic term “gene trap” is therefore used to refer to them collectively, regardless of the specific reporter gene construct. A system that allows gene activity to be monitored by creating gene fusions with a reporter gene was first used in bacterial genetics >20 years ago (Casadaban and Cohen, 1979). Random insertions of a lacZ reporter gene into the Escherichia coli chromosome were generated. These created gene fusions that could be used to monitor the expression of individual genes. In this way, genes could be identified based on their pattern of expression over time or under a variety of conditions in the absence of a mutant phenotype or any sequence information. Because the sequence of the inserted DNA was known, it provided a “tag” for easy isolation of the chromosomal gene. This approach has been modified for use in a number of species and has been extensively exploited in Drosophila and mouse genetics (reviewed in Bellen, 1999). Reporter genes can be used to construct three basic types of gene trap: enhancer trap, promoter trap, and gene trap (Figure 1). Each type is able to respond to cis-acting regulatory sequences at the site of insertion. In an enhancer trap (Figure 1B), the reporter gene is fused to a minimal promoter, typically containing a TATA box and transcription start site, that is unable to drive reporter gene expression alone but can be activated by neighboring enhancer elements. Promoter traps and gene traps contain a promoterless reporter gene so that expression can occur only when the insertion is within a transcriptional unit and in the correct orientation (Figures 1C and 1D). Expression of a promoter trap reporter gene requires that it be inserted into an exon, leading to a transcriptional fusion (Figure 1C). In contrast, gene trap constructs contain one or more splice acceptor sequences preceding the reporter gene (Figure 1D), which allow expression if insertion occurs in an intron. Splicing from the splice donor sites in the chromosomal gene to the splice acceptor sites in the reporter gene results in fusion of upstream exon sequences to the reporter gene. In addition to transcriptional fusions, promoter trap and gene trap insertions can also create translational fusions, which may provide information about protein localization. Structure of Enhancer, Gene, and Promoter Trap Vectors. (A) A generic chromosomal gene with exons (boxes) and introns (lines). (B) Enhancer trap construct. The minimal promoter of the reporter gene (TATA) is activated by a chromosomal enhancer element, resulting in expression of the reporter gene. (C) Promoter trap construct. The promoterless reporter gene can be expressed when insertion occurs in an exon so as to result in a transcriptional fusion. (D) Gene trap construct. The promoterless reporter gene contains splice acceptor (SA) sequences. Expression of the reporter gene occurs upon its insertion into an intron. Splicing from the chromosomal splice donor (SD) site to the SA sequence results in creation of a transcriptional fusion. Arrows in each panel represent the transcripts that are produced as a consequence of insertion. Each type of reporter gene construct has its own advantages. Because enhancer traps do not have the same constraints on expression as promoter and gene traps, which must insert within a gene and in the correct orientation, enhancer trap insertions lead to a higher frequency of reporter gene expression. However, expressed promoter or gene traps are more likely to cause gene disruption than are expressed enhancer traps. Also, because enhancers can activate gene expression at considerable distances, the genes controlling reporter gene expression may be more easily identified in promoter or gene trap insertions than in enhancer trap insertions. Gene traps provide a powerful tool for gene identification. Genes are identified based on reporter gene expression; therefore, a mutant phenotype is not required. This advantage allows identification of two classes of genes that are not easily amenable to classic genetic functionally redundant genes and genes that have functions at multiple developmental The identification of mutations in two redundant genes has in only been and et al., and mutations in redundant genes have by In disruption of a gene result in a phenotype that could However, if a gene trap insertion a the expression pattern the type of phenotype to for and may lead to of phenotypes that could be the other a in a gene that is at multiple stages of is likely to result in genes may result in phenotypes that are to approach Gene traps allow genes to be identified based on expression so that mutations are not a gene trap insertion may gene the disruption is not for gene identification. Therefore, functionally redundant genes expression patterns screening can be identified in the absence of an easily recognizable phenotype. genes can be identified based on reporter gene in if disruption genes that have at multiple developmental stages can be identified based on if mutations occur within genes. use gene traps, a of is that contains a reporter gene into the for reporter gene expression have been Each insertion is as a which can be for reporter gene expression. can be designed to identify genes that are expressed in specific cells or at specific developmental or in to an enhancer trap system was first in Drosophila years ago and The used to the reporter lacZ gene into the The lacZ gene was by the promoter, so that its expression could not be by for of this construct into the many different and lacZ expression that cis-acting regulatory could be this approach. systems used than to containing insertions and a for isolation of DNA et al., et al., et al., et al., about the same traps were in et al., et al., enhancer and promoter traps were et al., Because enhancers can over distances, it has been that the genes with an enhancer be more to identify with enhancer traps than promoter or gene traps This for with and low gene this in mouse has to use promoter or gene trap Gene trap constructs are typically into cells by a cells that for integration are to the mouse The gene trap used in mouse contains a lacZ reporter gene with one or more splice acceptor (SA) sequences (reviewed in and and 1999). insertion occurs in an will create a transcriptional fusion the exon sequence and In use of SA sequences the frequency of insertions that result in reporter gene expression by to et al., The gene trap systems in were designed to how insertions into genes. were by with a containing a promoterless gene to one et al., et al., of a plant on the of a gene fusion that to expression of the gene. 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