Abstract
Enhancer trapping (ET) is a powerful approach to establish tissue- or cell-specific reporters and identify expression patterns of uncharacterized genes. Although a number of enhancer-trapping vectors have been developed and a large library of fish lines with distinct tissue- or cell-specific expression of reporter genes have been generated, the specificity and efficiency of trapping vectors need to be improved because of the bias interaction of minimal promoters with genomic enhancers. Accordingly, we generated an enhancer-trapping vector pTME that contains a minimal mouse metallothionein gene (mMTI) promoter upstream of EGFP reporter. In the first round of screening, twelve zebrafish lines that carry a single copy of ET cassettes were characterized to have tissue- or cell-specific EGFP expression. One of the highly conserved noncoding elements near an insertion site of trapping cassettes was characterized as an enhancer that can specifically regulate the expression of EGFP in cells of the central nervous system. In addition, the pTME vector contains a mutation-cassette that is able to effectively block the transcription of an endogenous gene in an ET line with ubiquitous EGFP expression. Thus, the pTME vector can be used as an alternative tool for both enhancer trapping and mutagenesis across a target genome.
Highlights
Enhancer trapping (ET) is an effective approach widely used to characterize enhancers that control spatio-temporal expression patterns of genes in cells
We found that embryos injected with PTRE/G2 vector containing mouse metallothionein gene (mMTI) minimal promoter exhibited the weakest EGFP expression in comparison with those injected with vectors containing other minimal promoters (Fig 1B and 1C)
Levels of EGFP were detected by quantitative RT-PCR and the results verified that the mMTI exhibited the lowest expression activity than other three minimal promoters (Fig 1D)
Summary
Enhancer trapping (ET) is an effective approach widely used to characterize enhancers that control spatio-temporal expression patterns of genes in cells. A basic enhancer-trapping vector is composed of a reporter gene under the control of a minimal promoter [1]. Enhancers in the chromosome cannot drive the expression of a reporter gene in the trapping construct by themselves unless they induce the activity of a minimal promoter [2]. Since the activity of enhancers in a genome is able to regulate the transcription of endogenous genes, the distribution of a reporter in ET cassette can mimic the expression pattern of an endogenous gene and makes the ET approach effective to monitor gene activity throughout the genome [3].
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