Stable nuclear transformation of Pandorina morum.

BACKGROUND: Volvocine green algae like Pandorina morum represent one of the most recent inventions of multicellularity diverged from their unicellular relatives. The 8-16 celled P. morum alga and its close multicellular relatives constitute a model lineage for research into cellular differentiation, morphogenesis and epithelial folding, sexual reproduction and evolution of multicellularity. Pandorina is the largest and most complex organism in the volvocine lineage that still exhibits isogamous sexual reproduction. So far, molecular-biological investigations in P. morum were constricted due to the absence of methods for transformation of this species, which is a prerequisite for introduction of reporter genes and (modified) genes of interest. RESULTS: Stable nuclear transformation of P. morum was achieved using chimeric constructs with a selectable marker, a reporter gene, promoters and upstream and downstream flanking sequences from heterologous sources. DNA was introduced into the cells by particle bombardment with plasmid-coated gold particles. The aminoglycoside 3'-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus under control of an artificial, heterologous promoter was used as the selectable marker. The artificial promoter contained a tandem arrangement of the promoter of both the heat shock protein 70A (hsp70A) and the ribulose-1,5-bisphosphat-carboxylase/-oxygenase S3 (rbcS3) gene of Volvox carteri. Due to the expression of aphVIII, transformants gained up to 333-fold higher resistance to paromomycin in comparison to the parent wild-type strain.The heterologous luciferase (gluc) gene of Gaussia princeps, which was previously genetically engineered to match the nuclear codon usage of Chlamydomonas reinhardtii, was used as a co-transformed, unselectable reporter gene. The expression of the co-bombarded gluc gene in transformants and the induction of gluc by heat shock were demonstrated through bioluminescence assays. CONCLUSION: Stable nuclear transformation of P. morum using the particle bombardment technique is now feasible. Functional expression of heterologous genes is achieved using heterologous flanking sequences from Volvox carteri and Chlamydomonas reinhardtii. The aphVIII gene of the actinobacterium S. rimosus can be used as a selectable marker for transformation experiments in the green alga P. morum. The gluc gene of the marine copepod G. princeps, expressed under control of heterologous promoter elements, represents a suitable reporter gene for monitoring gene expression or for other applications in P. morum.

Stable nuclear transformation of Eudorina elegans.

BACKGROUND: 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 (Chlamydomonas reinhardtii) and a multicellular alga with differentiated cell types (Volvox carteri). Species with intermediate characteristics also exist, which blur the boundaries between unicellularity and differentiated multicellularity. These species include the globular alga Eudorina elegans, which is composed of 16-32 cells. However, detailed molecular analyses of E. elegans require genetic manipulation. Unfortunately, genetic engineering has not yet been established for Eudorina, and only limited DNA and/or protein sequence information is available. RESULTS: Here, we describe the stable nuclear transformation of E. elegans 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 (aphVIII) gene of Streptomyces rimosus, an actinobacterium, under the control of an artificial promoter consisting of two V. carteri 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 (gluc) gene from the marine copepod Gaussia princeps, which previously was engineered to match the codon usage of C. reinhardtii, was used as a reporter gene. The expression of gluc was mediated by promoters from C. reinhardtii and V. carteri. 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 gluc gene was demonstrated by transcription analysis and bioluminescence assays. CONCLUSIONS: Heterologous flanking sequences, including promoters, work in E. elegans and permit both constitutive and inducible expression of heterologous genes. Stable nuclear transformation of E. elegans is now routine. Thus, we show that genetic engineering of a species is possible even without the resources of endogenous genes and promoters.

Stable nuclear transformation of Gonium pectorale.

BACKGROUND: Green algae of the family Volvocaceae are a model lineage for studying the molecular evolution of multicellularity and cellular differentiation. The volvocine alga Gonium is intermediate in organizational complexity between its unicellular relative, Chlamydomonas, and its multicellular relatives with differentiated cell types, such as Volvox. Gonium pectorale consists of approximately 16 biflagellate cells arranged in a flat plate. The detailed molecular analysis of any species necessitates its accessibility to genetic manipulation, but, in volvocine algae, transformation procedures have so far only been established for Chlamydomonas reinhardtii and Volvox carteri. RESULTS: Stable nuclear transformation of G. pectorale was achieved using a heterologous dominant antibiotic resistance gene, the aminoglycoside 3'-phosphotransferase VIII gene (aphVIII) of Streptomyces rimosus, as a selectable marker. Heterologous 3'- and 5'-untranslated flanking sequences, including promoters, were from Chlamydomonas reinhardtii or from Volvox carteri. After particle gun bombardment of wild type Gonium cells with plasmid-coated gold particles, transformants were recovered. The transformants were able to grow in the presence of the antibiotic paromomycin and produced a detectable level of the AphVIII protein. The plasmids integrated into the genome, and stable integration was verified after propagation for over 1400 colony generations. Co-transformants were recovered with a frequency of approximately 30-50% when cells were co-bombarded with aphVIII-based selectable marker plasmids along with unselectable plasmids containing heterologous genes. The transcription of the co-transformed, unselectable genes was confirmed. After heterologous expression of the luciferase gene from the marine copepod Gaussia princeps, which was previously engineered to match the codon usage in C. reinhardtii, Gonium transformants show luciferase activity through light emission in bioluminescence assays. CONCLUSION: Flanking sequences that include promoters from C. reinhardtii and from V. carteri work in G. pectorale and allow the functional expression of heterologous genes, such as the selectable marker gene aphVIII of S. rimosus or the co-transformed, codon-optimized G. princeps luciferase gene, which turned out to be a suitable reporter gene in Gonium. The availability of a method for transformation of Gonium makes genetic engineering of this species possible and allows for detailed studies in molecular evolution using the unicellular Chlamydomonas, the 16-celled Gonium, and the multicellular Volvox.