Find in Library

<p>Abstract</p> <p>Background</p> <p>A fundamental step in evolution was the transition from unicellular to differentiated, multicellular organisms. Volvocine algae have been used for several decades as a model lineage to investigate the evolutionary aspects of multicellularity and cellular differentiation. There are two well-studied volvocine species, a unicellular alga (<it>Chlamydomonas reinhardtii</it>) and a multicellular alga with differentiated cell types (<it>Volvox carteri</it>). Species with intermediate characteristics also exist, which blur the boundaries between unicellularity and differentiated multicellularity. These species include the globular alga <it>Eudorina elegans</it>, which is composed of 16–32 cells. However, detailed molecular analyses of <it>E. elegans</it> require genetic manipulation. Unfortunately, genetic engineering has not yet been established for <it>Eudorina</it>, and only limited DNA and/or protein sequence information is available.</p> <p>Results</p> <p>Here, we describe the stable nuclear transformation of <it>E. elegans</it> by particle bombardment using both a chimeric selectable marker and reporter genes from different heterologous sources. Transgenic algae resistant to paromomycin were achieved using the aminoglycoside 3^′-phosphotransferase VIII (<it>aph</it>VIII) gene of <it>Streptomyces rimosus</it>, an actinobacterium, under the control of an artificial promoter consisting of two <it>V. carteri</it> promoters in tandem. Transformants exhibited an increase in resistance to paromomycin by up to 333-fold. Co-transformation with non-selectable plasmids was achieved with a rate of 50 - 100%. The luciferase (<it>gluc</it>) gene from the marine copepod <it>Gaussia princeps</it>, which previously was engineered to match the codon usage of <it>C. reinhardtii</it>, was used as a reporter gene. The expression of <it>gluc</it> was mediated by promoters from <it>C. reinhardtii</it> and <it>V. carteri</it>. Heterologous heat shock promoters induced an increase in luciferase activity (up to 600-fold) at elevated temperatures. Long-term stability and both constitutive and inducible expression of the co-bombarded <it>gluc</it> gene was demonstrated by transcription analysis and bioluminescence assays.</p> <p>Conclusions</p> <p>Heterologous flanking sequences, including promoters, work in <it>E. elegans</it> and permit both constitutive and inducible expression of heterologous genes. Stable nuclear transformation of <it>E. elegans</it> is now routine. Thus, we show that genetic engineering of a species is possible even without the resources of endogenous genes and promoters.</p>