Abstract
Although transposon-mediated enhancer trapping (ET) is successfully applied in diverse models, the efficiency of various transposon systems varies significantly, and little information is available regarding efficiency of enhancer trapping by various transposons in zebrafish. Most potential enhancers (Ens) still lack evidence of actual En activity. Here, we compared the differences in ET efficiency between sleeping beauty (SB), piggyBac (PB) and Tol2 transposons. Tol2 represented the highest germline transfer efficiencies at 55.56% (NF0 = 165), followed by SB (38.36%, NF0 = 151) and PB (32.65%, NF0 = 149). ET lines generated by the Tol2 transposon tended to produce offspring with a single expression pattern per line, while PB and SB tended to generate embryos with multiple expression patterns. In our tests, 10 putative Ens (En1–10) were identified by splinkerette PCR and comparative genomic analysis. Combining the GFP expression profiles and mRNA expression patterns revealed that En1 and En2 may be involved in regulation of the expression of dlx1a and dlx2a, while En6 may be involved in regulation of the expression of line TK4 transgene and rps26, and En7 may be involved in the regulation of the expression of wnt1 and wnt10b. Most identified Ens were found to be transcribed in zebrafish embryos, and their regulatory function may involve eRNAs.
Highlights
The term ‘enhancer’ (En) was first introduced to describe the effects of SV40 DNA on the ectopic expression of the gene for a cloned rabbit β-globin [1]
To compare the transposition frequency of three transposons in zebrafish embryos, we co-injected a mixture of enhancer trapping (ET) vector and transposase mRNA of PB, sleeping beauty (SB), or Tol2 transposon into one-cell stage embryos of zebrafish, and screened for green fluorescent protein (GFP) expression at 48 and 120 hpf
We found that the Tol2 transposon represented the highest GFP expression (90.45%, 92.71%, N = 1524), followed by SB
Summary
The term ‘enhancer’ (En) was first introduced to describe the effects of SV40 DNA on the ectopic expression of the gene for a cloned rabbit β-globin [1]. Ens can mediate the on/off switch patterns of gene expression in specific cell types at particular stages during animal embryogenesis and development [2]. Ens can act at a long distance away from their target gene and independent of their orientation [3]; some Ens have been shown to be involved in genetic disease in humans [4,5]. These functional properties make Ens an important part of the genomic regulatory architecture. As the development of retrovirusGenes 2018, 9, 630; doi:10.3390/genes9120630 www.mdpi.com/journal/genes
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