Swapped green algal promoters: aphVIII-based gene constructs with Chlamydomonas flanking sequences work as dominant selectable markers in Volvox and vice versa
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Production of transgenic organisms is a well-established, versatile course of action in molecular biology. Genetic engineering often requires heterologous, dominant antibiotic resistance genes that have been used as selectable markers in many species. However, as heterologous 5′ and 3′ flanking sequences often result in very low expression rates, endogenous flanking sequences, especially promoters, are mostly required and are easily obtained in model organisms, but it is much more complicated and time-consuming to get appropriate sequences from less common organisms. In this paper, we show that aminoglycoside 3′-phosphotransferase gene (aphVIII) based constructs with 3′ and 5′ untranslated flanking sequences (including promoters) from the multicellular green alga Volvox work in the unicellular green alga Chlamydomonas and flanking sequences from Chlamydomonas work in Volvox, at least if a low expression rate is compensated by an enforced high gene dosage. This strategy might be useful for all investigators that intend to transform species in which genomic sequences are not available, but sequences from related organisms exist.
KeywordsGreen algae Volvocales Recombinant DNA technology Transgenic expression Genetic transformation Paromomycin resistance
Aminoglycoside 3′-phosphotran sferase (VIII) gene
Heat shock protein 70A ge ne
Ribulose-1,5-bisphosphat-carboxylase small subunit gene 2 from Chlamydomonas reinhardtii
Ribulose-1,5-bisphosphat-carboxylase small subunit gene 3 from Volvox carteri
We wish to thank Irina Sizova and Peter Hegemann for providing plasmid pSI103, Thomas Jakobiak and Rüdiger Schmitt for providing plasmid pPmr3, and Kordula Puls for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 521).
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