Progress 09/15/06 to 09/14/09
Outputs
OUTPUTS: The overall goal of this project is to produce a rotavirus vaccine in chloroplasts of sugarcane. The inverted repeat region of the sugarcane chloroplast DNA was identified and incorporated into the chloroplast transformation vectors. Several selectable marker expression cassettes (including aadA, confers resistance to spectinomycin and streptomycin; aphA-6, confers resistance to kanamycin and amikacin; nptII, confers resistance to kanamycin; hpt, confers resistance to hygromycin) were constructed. A rotaviral cDNA whose transcription is driven by highly active chloroplast gene promoters, translational regulatory and RNA stability regions were also incorporated into the transformation vectors. Multiple bombardments were carried out to evaluate the transformation conditions, such as biolistic delivery pressure, antibiotic selection level and interval. Several experiments of transformation using chloroplast transformation vectors were conducted via particle bombardment of embryonic calli, derived from a commercial sugarcane variety, H65-4671. The bombarded embryonic calli were selected using kanamycin or hygromycin for at least three cycles with a one-month interval. The selected embryogenic calli survived on the selection medium were micropropagated for obtaining materials for further analysis. PCR using primers based on the inverted repeat region and selectable markers were carried out, however, the PCR results were inconclusive due to either non-transformed escapes resulted from low selection pressure or the heteroplasmic nature of the chloroplast population due to low copies of transgene. We have also used the project in a demonstration for the Advances in Biosciences Education workshop for community college faculty and students in Hawaii. PARTICIPANTS: Dr. Christen Y. Yuen, Post-doctoral Research Associate, Dept. Molecular Biosciences & Bioengineering, University of Hawaii Dr. David Christopher, Professor, Dept. Molecular Biosciences & Bioengineering, University of Hawaii Dr. Y. Judy Zhu, Hawaii Agricultural Research Center, Aiea, Hawaii TARGET AUDIENCES: Plant and medical biologists and industries interested in safe vaccine development. All scientists interested in keeping transgenes and GMOs out of pollen. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Genetically engineered plants have great potential in agriculture to reduce the use of harmful pesticides and to produce high value compounds in safer and economical ways. However, perceived risks associated with genetic modification have led to restrictions, lawsuits and rejection of genetically altered crops. Hence, some of the important benefits that these crops offer to society may go unrealized. One major concern was that modified DNA will escape into the environment through pollen. In this project, a chloroplast transformation was attempted to produce human vaccines in sugarcane chloroplasts, which will reduce the environmental risk associated with dispersal of genetically modified DNA in pollen. The vaccine chosen was for rotavirus, which causes widespread infectious gastroenteritis in infants and farm animals. The rotavirus subunit antigen employed here is a safe an effective vaccine for immunization against the disease, however production by traditional systems is limited. Moreover, a previously developed vaccine made from attenuated whole virus has been discommended. Therefore, there is a need for large-scale production of a safe subunit vaccine. Sugarcane was chosen because the highest levels of photosynthetic productivity of any plant have been measured in sugarcane in Hawaii. This productivity and the strong protein synthesizing capacity of chloroplasts are expected to enhance vaccine production. The chloroplast transformation system which we attempt to develop in this work can also be expanded to produce other high value compounds in sugarcane.
Publications
- No publications reported this period
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: Several kanamycin and hygromycin resistant lines are being screened at the calli level. However, none are streptomycin resistant, meaning that the transgene may not have entered the chloroplast. The main outputs are the final characterization of these lines for the presence of the rotaviral transgene and production of vaccine protein PARTICIPANTS: Chris Yuen, post-doctoral scientist University of Hawaii David Christopher, professor, University of Hawaii Y. Judy Zhu, Hawaii Agricultural Research Center TARGET AUDIENCES: Scientists working on chloroplast molecular biology in industry and pharmaceutical companies working on vaccine development. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This new technology will boost production of rotavirus vaccine that is stable and does not require refrigeration. Traditional production methods of the rotavirus vaccine have been problematic and have given poor yields. The vaccine produced in sugarcane plants is safe as a human vaccine for use in immunization of infants against dysentery, a life-threatening disease of the tropics. In addition, the vaccine can be used for protection of farm animals. Furthermore, the use of chloroplast transformation will prevent the spread of bioengineered genes in the pollen, so sugarcane can be grown large-scale in the field. The vaccine-producing sugarcane plants will provide a valuable and safe alternative crop for Hawaii agriculture.
Publications
- No publications reported this period
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Progress 09/15/06 to 09/14/07
Outputs
OUTPUTS: Because of competition with a research group in Australia, we have not publicly discussed our results and outputs. We have used the project in a demonstration for the Advances in Biosciences Education workshop for community college faculty and students in Hawaii. We described how the method of chloroplast transformation is being applied to sugarcane to express a vaccine protein against the dysentery-causing rotavirus. Chloroplast transformation is attractive because it will generate high levels of vaccine protein and does not lead to the spread of genetically engineered DNA into pollen. Since chloroplast transformation has not been reported in sugarcane, it is being developed from inception in this project. The inverted repeat region of the sugarcane chloroplast DNA was identified and incorporated into the chloroplast transformation vectors. Selectable marker expression cassettes (aadA, confers resistance to spectinomycin and streptomycin); aphA-6, confers resistance to
kanamycin and amikacin; nptII, confers resistance to kanamycin; hpt, confers resistance to hygromycin), a rotaviral cDNA whose transcription is driven by highly active chloroplast gene promoters, translational regulatory and RNA stability regions were also added to the vector. Chloroplast transformation is being conducted via particle bombardment of embryonic calli.
PARTICIPANTS: David A. Christopher (professor, principal investigator), University of Hawaii, CTAHR, Dept. MBBE. Yun Judy Zhu (co-principal investigator), Hawaii Agricultural Research Center, Affiliated graduate faculty MBBE. Christen Yuen (post-doctoral junior researcher), University of Hawaii, CTAHR, Dept. MBBE.
TARGET AUDIENCES: American Society of Plant Biologists, NATO, United Nations
Impacts
This technology will help increase the number of doses of safe vaccine available to decrease the incidence of rotaviral-induced dysentery. Traditional production methods of the rotavirus vaccine have been problematic and have given poor yields. The new approach outlined in this project involves expression of a rotaviral coat protein antigen in sugarcane chloroplasts. This will allow maximal production of a safe supply of this human vaccine for use in immunization of infants against dysentery, a life-threatening disease of the tropics. In addition, the vaccine can be used for protection of farm animals. Furthermore, the use of chloroplast transformation will prevent the spread of bioengineered genes in the pollen, so sugarcane can be grown large-scale in the field. The vaccine-producing sugarcane plants will provide a valuable and safe alternative crop for Hawaii agriculture.
Publications
- No publications reported this period
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