title: Expression levels of domestic cDNA cassettes integrated in the nuclear genomes of various Chlamydomonas reinhardtii strains.
abstract: We attempted to overexpress three types of expression cassettes, each of which contained a different open reading frame (ORF) of domestic Chlamydomonas cDNAs. Each ORF was strongly driven by an artificial hybrid promoter. We used two wild-type Chlamydomonas strains (i.e., CC-124 and CC-125) and two mutant strains [i.e., UV-mutated (UVM) 4 and UVM11] that have been reported to have a high potency for expressing nondomestic nuclear transgenes. We found that the 1-deoxy-d-xylulose-5-phosphatesynthase (DXS1), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR1), and squalene synthase (SQS) cassettes were not readily overexpressed in the wild-type strains at levels where the products were clearly detectable by Western blotting using a monoclonal antibody. In contrast, Western blot-positive SQS cassette transformants were frequently detected in the UVM4 and UVM11 strains, i.e., at an approximately 4.5 times higher frequency than that in the CC-124 wild-type strain. Moreover, transformants that accumulated large amounts of the SQS protein were obtained frequently in the UVM4 and UVM11 strains, i.e., the frequency was approximately 2.2 times higher than that in the CC-124 strain. However, a position effect of the integrated expression cassette was obviously detected not only in the wild-type but also in UVM strains. This suggests that the epigenetic repression mechanism of transgenic genes was not completely knocked out, even in the UVM strains. Further improved Chlamydomonas strains are essential to facilitate high-throughput screening of transformants that express nuclear transgenes at a high level.
authors.affiliation: School of Environmental Science and Engineering, Kochi University of Technology (KUT), Tosayamada, Kochi 782-8502, Japan.
authors.forename: Fantao
authors.initials: F
authors.lastname: Kong
authors.affiliation: School of Environmental Science and Engineering, Kochi University of Technology (KUT), Tosayamada, Kochi 782-8502, Japan.
authors.forename: Tomohito
authors.initials: T
authors.lastname: Yamasaki
authors.affiliation: School of Environmental Science and Engineering, Kochi University of Technology (KUT), Tosayamada, Kochi 782-8502, Japan; Japan Science and Technology Agency, CREST, 5, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan. Electronic address: ohama.takeshi@kochi-tech.ac.jp.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Ohama
mesh_terms: D002467:Cell Nucleus / Q000235:genetics*; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D016678:Genome / Q000235:genetics*; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics
chemical_list: D018076:DNA, Complementary
keywords: Chlamydomonas reinhardtii; Nuclear transformation; Overexpression; UV-mutated strains; cDNA expression cassettes