Abstract
The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimentally express genes in specific cells of an organism. We recently established a first transgenic strain of the annelid Platynereis, using a Tc1/mariner-type Mos1 transposon vector. Here, we compare Mos1 with Tol2, a member of the hAT family of transposons. In Platynereis, Tol2-based constructs showed a higher frequency of nuclear genome insertion and sustained gene expression in the G0 generation. However, in contrast to Mos1-mediated transgenes, Tol2-mediated insertions failed to retain fluorescence in the G1 generation, suggesting a germ line-based silencing mechanism. Furthermore, we present three novel expression constructs that were generated by a simple fusion-PCR approach and allow either ubiquitous or cell-specific expression of a reporter gene. Our study indicates the versatility of Tol2 for transient transgenesis, and provides a template for transgenesis work in other emerging reference species.
Highlights
The selection of a few species as ‘‘molecular model systems’’ has helped researchers to gain deep insight into the molecular processes underlying cell biology and development of these organisms
In Platynereis, meganuclease has so far only led to mosaic expression of a tested reporter (Kristin Tessmar-Raible, unpublished), but in fishes, amphibians, ascidians, or cnidarians, meganuclease-mediated transgenesis is an alternative to transposon-based approaches
Similar to Tol2, we observe genomic integration of Mos1-based constructs Together with the excision assay, these findings indicate that Mos1 cleaves co-injected DNA constructs, and that these constructs can integrate into the Platynereis genome, even though excision and/or transposition mechanisms seem to occur with reduced efficiency, and expression rates are consistently lower than the ones observed for Tol2-mediated transgenesis
Summary
The selection of a few species as ‘‘molecular model systems’’ has helped researchers to gain deep insight into the molecular processes underlying cell biology and development of these organisms. It has been argued that biologists need to start selecting and exploring new model systems [1,2]. One step into this direction has been enabled by the development of cost-effective massive sequencing technology. This technology has facilitated the deciphering of an increasing number of genomes and transcriptomes of potential new model systems. [7,8]), and serve as a basis for the comparison of gene expression patterns with and between emerging model species, thereby providing insight into the evolution of tissues and cell types Systematic gene expression data have been generated for conventional model species (see e.g. refs. [7,8]), and serve as a basis for the comparison of gene expression patterns with and between emerging model species, thereby providing insight into the evolution of tissues and cell types (reviewed in ref. [9])
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