Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system.
The successful expression of foreign genes mainly depends on both a reliable method for transformation and a suitable promoter sequence. We created a series of modular plasmids that facilitate the rapid construction of large tandem vectors for transgene expression under the control of different promoter sequences in Chlamydomonas reinhardtii. Tandem vectors carrying expression cassettes for Renilla luciferase and a metabolic selection marker (ARG7) were manufactured by fusing two plasmids in vitro using Cre/lox site-specific recombination. Supercoiled and linear plasmids were used to transform an arginine auxotrophic Chlamydomonas strain, and rates of co-expression as well as levels of luciferase activity were monitored for frequently used promoters (HSP70A, LHCB1, PSAD, and the chimeric HSP70A/RBCS2). Linearized tandem vectors generally increased the co-expression frequency (up to 77%) compared with standard cotransformation protocols. Most transformants showed a single and complete integration event confirming the close linkage of active selectable marker and reporter gene within the nuclear genome. The analysis of luciferase activity showed expression levels within three orders of magnitude for the promoters used, with the artificial HSP70A/RRBCS2 being the most active. For 69% of all luminescent transformants carrying the HSP70A promoter luciferase expression was enhanced by heatshock, indicating physiological promoter function in a transgenic context.
Heitzer
Markus
M
University of Regensburg, Heidelberg, Center of Excellence for Fluorescent Bioanalysis, Germany. markus.heitzer@exfor.uni-regensburg.de
Zschoernig
Barbara
B
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; C045073:Cre recombinase; D019426:Integrases