Chloroplasts are organelles present in photosynthetic plant cells. Their principal function is to capture energy from light to fix atmospheric CO2 and convert it into sugars. They possess their own genome with a variable size up to several hundred kilobases; each chloroplast can contain up to 100 copies of its own genome. The number of chloroplast varies between 1 and more than 100 in higher plants.
Existing technologies for chloroplast transformation use particle bombardment delivery of DNA into organelles. The major restriction of this approach however is the low efficiency of DNA targeting into organelles and the requirement for several rounds of tissue culture on selective media to achieve homoplasmy.
Algentech’s proprietory chloroplast transformation technology does not rely on particle bombardment for passive delivery of DNA into chloroplasts but instead it is using genetic units constitutively expressed from the nucleus as an unlimited source of molecules that can be targeted into these organelles. The chloroplast transformation machinery is composed of three elements, expressed in the cytoplasm from the nucleus under strong constitutive promoters:
(i) a chloroplast transformation cassette composed of left and right flanking sequences (LFS and RFS) homologous to a specific location on the chloroplast genome used to facilitate insertion of DNA sequence of interest into the chloroplast genome using homologous recombination, a chloroplast expression cassette, a sequence recognised by the translocation protein and a priming sequence for reverse transcription of RNA into cDNA
ii) a translocation protein with high affinity to the chloroplast transformation cassette used to facilitate the transfer of RNA molecules of interest into chloroplasts;
(iii) a protein with reverse transcriptase activity targeted into the chloroplast stroma used for reverse transcription of the chloroplast transgene cassette into cDNA that serves as a template for homologous recombination at specific sites in the chloroplast genome.
Chloroplast transformation technologies were developed to transform these organelles into cell factories for production of molecules of high commercial value (therapeutic compounds, recombinant proteins, precursors for biofuel production). These technologies use the repetition of the chloroplast genetic material (up to 10,000 copies of the genome per cell) to produce high amounts of protein. Moreover, chloroplasts are not usually transmitted by pollen (maternal inheritance), therefore the risk of transfer of modified genes to other plants are minimal.
In the pipe:
We are currently developing new tools for transient expression of genetic units required for chloroplast transformation in order to avoid nuclear transgene insertion. We are also working on improvements to achieve homoplasmy after the first round of transformation.