Progress 10/01/06 to 09/30/07
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 tot he 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; National Agricultural Biotechnology Council
Impacts
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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Progress 09/15/04 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 tot he 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; National Agricultural Biotechnology Council
Impacts
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. Please note: Research will continue under Project HAW00561-06G
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
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 tot he vector.
Chloroplast transformation is being conducted via particle bombardment of embryonic calli.
Impacts
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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Progress 10/01/04 to 09/30/05
Outputs
The research supports efforts to transform specific rotaviral coat protein cDNAs into sugarcane chloroplasts via homologous recombination. Chloroplast transformation has been used by researchers to exploit the high protein production capacity of the intracellular compartment and to avoid transmission of the added transgenes to pollen. However, chloroplast transformation has not been reported in sugarcane. The inverted repeat region of the sugarcane chloroplast DNA has been recently elucidated, and the appropriate integration site and other regulatory regions have been identified and amplified for the construction of chloroplast transformation vectors. The coPI (JZ) has established a highly efficient nuclear genetic transformation system and tissue culture and regeneration protocol for sugarcane. Other efforts during the past year have emphasized construction of an effective chloroplast transformation vector. Versions of this vector under construction contain a
selectable marker expression cassette and a rotaviral cDNA whose transcription is driven by the psbA and 16S rrn promoters, a 5' leader from the psbA gene, and a 3' untranslated region from psbC gene will stabilize the transcript. The following selectable markers have been incorporated into this cassette: aadA (confers resistance to spectinomycin and streptomycin); aphA-6 (confers resistance to kanamycin and amikacin); nptII (confers resistance to kanamycin) and hpt (confers resistance to hygromycin). Chloroplast transformation is achieved via particle bombardment, and the PIs are in the process of standardizing particle gun parameters for nuclear transformation. The most suitable particle gun parameters for nuclear transformation will be used for chloroplast transformation.
Impacts
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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Progress 10/01/03 to 09/30/04
Outputs
The overall goal of this project is to produce a rotavirus vaccine in chloroplasts of sugarcane. The researchers will construct gene shuttle vectors for delivering the rotavirus vaccine cDNA and various reporter genes into sugarcane chloroplasts. The sugarcane chloroplast genome will be genetically transformed and screened to verify the maternal inheritance of the transgenes. Homoplasmic sugarcane plants with the highest vaccine expression will be selected. The vaccine protein will be purified for immuno-activity assays. This project has just started in September, 2004. We had offered the junior researcher position to a highly qualified candidate who initially accepted, then decided not to come here. We continue to advertise and search. A technician in Dr. Christopehr's lab, Kristie Okazaki, has prepared some basic plasmid and chloroplast clones and vectors. Dr. Zhu and the PI have contacted the University of Florida to allow Dr. Zhu to travel there to conduct
sugarcane chloroplast transformation with the vectors in preparation.
Impacts
Production of a safe vaccine for rotavirus in sugarcane plants will provide an alternative use of sugarcane and strengthen diversified agriculture in Hawaii. Possible patents from this work will increase revenues into UH and CTAHR. Both will help the local economy. Increasing the supply of a rare vaccine could save thousands of infant lives in the tropics.
Publications
- No publications reported this period
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