Plant cells contain several compartments, including the nucleus, the mitochondria and the chloroplasts, where photosynthesis is conducted. Any and all of these components can be penetrated via the most widely used method of transforming cells, the patented gene gun. The gene gun, which Chlorogen and many other companies use to introduce new genes into plants, is not precise. It bombards a section of plant material with tiny metal particles coated with DNA. The process is just as likely to shoot new genetic material into the chloroplast as the nucleus or mitochondria.

However, with proper genetic instructions, the introduced genes will function only inside the chloroplast. Chlorogen has invented and patented genetic sequences or regulatory signals that tell foreign genes to function within the chloroplasts and only the chloroplasts. Research by Chlorogen and other groups has demonstrated successful chloroplast transformation in several crops.

Chloroplasts

Green plant cells contain three distinct genetic entities, a single nuclear genome and 10 to 100 plasmatic organelle genomes ⁽¹⁾. These organelles are the chloroplasts and the mitochondria, each with multiple copies of a circular genome. Each organelle contains replication, transcription, translation and processing machinery separate from the rest of the cell both functionally and physically. Since the late 1980s the light-harvesting chloroplast has been the focus of intense research for genetic engineering. The past 15 years of development have established that plants with transformed chloroplasts have potential to provide low-cost, stable and quality proteins ^(2,3,4).

Chloroplast Transformation/Expression Vector

The chloroplast transformation/expression vector (CTT™ vector) is designed to insert the transgene cassette into a defined site in the chloroplast genome. Using homologous chloroplast DNA sequences that flank the transgene cassette and the highly efficient chloroplast homologous recombination machinery, the transgene cassette is integrated in a specific orientation. Each transformed plant that is generated will have the exact same site of insertion and orientation of genes.
      The gene insertion site has been defined as a highly active transcription region of the chloroplast. The inserted genes are co-expressed at high levels in a polycistronic mRNA with a selectable marker gene. Chlorogen optimizes the gene sequence(s) and translational regulatory elements linked to each gene. For each protein, a set of optimized CTTª vectors is developed and evaluated.

Biolistic Mediated Gene Transfer

The CTT™ vector DNA is coated onto gold particles and delivered to the chloroplasts using biolistic mediated transfection.



      Once the transgene cassette has been integrated into a copy of the chloroplast genome and 20-25 cell divisions occur under selective conditions, then all chloroplasts within each cell will contain the transgene. Chloroplast genomes are maternally inherited, so all seeds from a single plant will contain the transgene. Therefore, the CTT™ system can produce master seed stock in a single generation.

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1. Palmer, JD 1985 Comparative organization of chloroplast genomes. Annu. Rev. Genet 19: 325-354.
2. Daniell H, McFadden BA. (1987) Uptake and expression of bacterial and cyanobacterial genes by isolated cucumber etioplasts. Proc Natl Acad Sci U S A. 84(18):6349-53. Erratum in: Proc Natl Acad Sci U S A 1988 Jan;85(1):93.
3. Ruf S, Hermann M, Berger IJ, Carrer H, Bock R. (2001) Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat Biotechnol.,19:870-5.
4. Sidorov VA, Kasten D, Pang SZ, Hajdukiewicz PT, Staub JM, Nehra NS. (1999) Technical Advance: Stable chloroplast transformation in potato: use of green fluorescent protein as a plastid marker. Plant J. 19:209-216.