reinhardtii includes numerous functionally uncharacterized genes. In cases in which transgenes were obtained using such vectors, the marker gene and transgene are genetically linked and usually inherited together. Several vectors systems were developed for this purpose, including those enabling sustained expression of transgenes in recipients. One strategy to increase co-transformation frequency is the use of a marker gene physically linked to a gene of interest. reinhardtii was not examined systematically, we presume that multiple plasmids are frequently integrated at the same locus, leading to a tight genetic linkage between marker genes and co-introduced transgenes in transgenic C. Thus, although the fate of multiple-plasmid co-transformation in C. Co-transformation of plants by Agrobacterium tumefaciens-mediated transformation using multiple plasmids also resulted in the integration of multiple T-DNAs at the same locus on plant chromosomes. Such tight linkage between a marker gene and co-introduced transgenes was observed in transgenic rice and soybean generated by biolistic bombardment, in which most of the transgenes were co-integrated together with a marker gene at one or multiple loci. However, this technique cannot be used if the linkage between a marker gene and a co-introduced transgene is tight. Sexual crossing is a powerful tool for this purpose. Therefore, efficient methods for the removal of marker genes from transgenic C. reinhardtii for various industrial applications, there are public concerns regarding the spread of marker genes in the environment. For the large-scale deployment of transgenic C. reinhardtii is considered to be a model organism for basic research and an industrial biotechnology host. reinhardtii is limited even though availability of multiple selectable markers is necessary for the sequential introduction of transgenes.Ĭ. However, the number of selectable marker genes used in C. Recently, this alga has also been used to manipulate metabolic pathways involved in biofuel and hydrogen production using the range of genetic manipulation tools available to this organism. The green unicellular alga Chlamydomonas reinhardtii has been widely used as a model system for studying the genetic and molecular mechanisms of biological processes such as photosynthesis and flagellar motility. reinhardtii could further increase the potential of this organism for use in basic and applied research. This precise Cre-mediated deletion method applicable to transgenic C. The ble-(linker)- CrCRE expression cassette remained in the genome after excision of the aphVIII expression cassette, and it was subsequently removed by crossing with the wild-type strain. Similarly, from strain cc124 carrying a single loxP-flanked aphVIII expression cassette, the aphVIII expression cassette was successfully excised after introduction of the ble-(linker)- CrCRE expression cassette. When the ble-(linker)- CrCRE expression cassette was integrated into the genome of strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette, CrCre recombinase-mediated excision of the aphVIII expression cassette was observed at a frequency higher than that in stable transformants of the CrCRE expression cassette. Therefore, the ble-(linker)- CrCRE fusion protein is expected to localize in the nucleus. The product of ble (Ble) is a bleomycin-binding protein that confers resistance to bleomycin-related antibiotics such as Zeocin and localizes in the nucleus. We then constructed an expression cassette of an in-frame fusion of ble to CrCRE via a short linker peptide.
Among 46 transformants carrying the CrCRE expression cassette stably, the excision of aphVIII by CrCre recombinase was observed only in one transformant. reinhardtii, was synthesized, and a CrCRE expression cassette was introduced into strain cc4350 carrying a single copy of the loxP-flanked aphVIII expression cassette. Separately, a synthetic Cre recombinase gene ( CrCRE), the codons of which were optimized for expression in C. reinhardtii strains cc4350 and cc124 carrying an aphVIII expression cassette flanked by two direct repeats of loxP were constructed. For the first time, we applied the Cre/ loxP-mediated site-specific recombination system to Chlamydomonas reinhardtii to construct marker-free transgenic strains. The Cre/ loxP system has particularly been used for the removal of selectable marker genes to create marker-free transgenic organisms. The Cre/ loxP system has been used to achieve targeted insertion and precise deletion in many animal and plant genomes.
The Escherichia coli bacteriophage P1 encodes a site-specific recombinase called Cre and two 34-bp target sites of Cre recombinase called loxP.
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