Progress 09/01/05 to 07/31/10
Outputs
OUTPUTS: To ameliorate some of the concerns associated with the release or escape of transgenic fish, a novel, non-sterility based biological containment method was devised that utilizes two site-specific recombination systems. In this system, the FLP-recombinase mediated removal of an FRT-flanked gene-of-interest triggers the Cre-recombinase mediated self-excision of the recombinase genes, leaving behind a single 34-bp loxP footprint. The recombinases in this strategy are driven by a germ cell-specific promoter such that only gametes excise the transgene, thus retaining the beneficial traits associated with the gene-of-interest in somatic tissue. To assay the efficiency of site-specific recombination in transgenic fish carrying a construct consisting of a green fluorescent protein (GFP) marker transgene flanked by both FRT and loxP sites, FLPe recombinase capped RNA was microinjected into single cell stage zebrafish embryos obtained by crossing hemizygous transgenic
males with wild-type females. By 48 hours post fertilization (hpf), the proportion of embryos displaying green fluorescence following FLPe RNA microinjection was significantly lower (7.7%; p<0.001) than expected (50%) if excision had not occurred. Embryos that retained fluorescence displayed marked mosaicism. Inheritance of the excised transgene in non-fluorescent, transgenic embryos was verified by PCR amplification of a recombinase-mediated excision product, and FLPe-mediated recombination was confirmed by DNA sequencing. When sperm samples from phenotypically mosaic fish were analyzed, individuals were identified where the sperm was also positive for a Cre-excision PCR product, indicating that the loxP-flanked transgene had also been excised from a portion of the germ cells. As a proof-of-concept, this showed that two site-specific recombinases could be used to sequentially excise transgenes, including the transgenes encoding the recombinases themselves, from the germline of
transgenic fish. We then tested this system in vivo by crossing separate lines of transgenic fish, each carrying a construct designed to remove transgenic DNA from the gametes of fish carrying a copy of both constructs. However, the germline transgene excision frequency observed in the double transgenic fish very low. Although the results of the flpe RNA microinjections appeared promising and offered a working proof-of-concept, ultimately the real world application of two germ cell-driven site-specific recombination systems for the genetic containment of transgenic fish was far too inefficient for this strategy to be a utilized as a means of genetic containment for transgenic fish. The results of this research project have been disseminated to the research community through conferences and publications in scientific journals, including a review paper entitled "Transgenic Approaches for the Reproductive Containment of Genetically Engineered Fish" that was published in the journal
Aquaculture in 2008. Additionally several general audience publications have resulted from this project including fact sheets. Finally, results were shared with research and regulators at the 2009 Transgenic Animal Conference. PARTICIPANTS: Individuals: Dr. Alison Van Eenennaam (Principal Investigator) set the framework for this project and provided guidance to graduate students to meet specific objectives outlined in the proposal. Andrew Wong was the graduate student who worked full time on this project and who designed and created all the constructs in this project. He tested various constructs in yeast before implementing them in zebrafish. He microinjected all the constructs into zebrafish and performed all the screening and genotyping. He completed his Ph.D. dissertation on this project. Partner Organizations: Aqua Bounty Technologies allowed Andrew Wong to participate in a 3-month internship at the Aqua Bounty Technologies in San Diego. Collaborators: Dr. Bruce Draper is an
Assistant Professor at UC Davis who is studying the development and maintenance of germline stem cells in zebrafish. Dr. Draper has setup a zebrafish rearing facility at UC Davis and provided expertise related to zebrafish rearing and transgenesis as a collaborator on this project. Training and professional development: This project provided training and professional development opportunities for a graduate student to undertake all of the experimental work, and understand the biology and genetics behind reproductive containment of transgenic fish, and the issues associated with their commercialization. Andrew Wong, the Ph.D. student who worked on this project, recently applied for a position with the FDA transgenic animal regulatory group. TARGET AUDIENCES: Research results were presented at the XVII International Plant & Animal Genome Meeting, Jan.10-14, 2009. San Diego, CA. The results of this project were presented to transgenic scientists and regulators charged with the regulation
of genetically engineered animals at the Transgenic Animal Research Conference VII. August 17-22, 2009. Tahoe City, CA. PROJECT MODIFICATIONS: As reported last year lack of gonadal fluorescent protein expression from the male line construct led us to try a new zebrafish germ cell-specific promoter, ziwi. Additionally, as we were having difficulty identifying transgenic founder fish using traditional transgenesis methods, we adopted a transposon-mediated transgenesis technique to increase our chances of obtaining germline positive transgenic founder fish. These two modifications delayed the completion of this project for an additional year.
Impacts
The development of fast-growing, transgenic fish that efficiently use resources (time, feed, etc.) and produce less waste could result in decreased costs for the aquaculturist and a more affordable, sustainable fish supply for consumers. However, the environmental risk associated with the escape and interbreeding of genetically engineered fish with wild populations is considered to be the greatest science-based concern facing the animal biotechnology industry. There is a need for biocontainment strategies to minimize the ecological risks of transgene flow from genetically engineered fish into native fish populations. This pilot study was designed to determine the efficacy of recombinase-mediated excision events to selectively eliminate transgenic DNA from the germ cells of genetically-engineered zebrafish. Although we were able to reduce our approach to practice, the low frequency of germline transgene excision events revealed that our approach to use two germ
cell-driven site-specific recombination systems was not sufficiently robust to guarantee containment genetic containment of transgenic fish should they escape into the wild and mate with native populations of fish. Other methods for biological or genetic containment of transgenic fish have also failed to achieve 100% containment. Currently it appears that, if complete containment is the goal, the most foolproof approach may involve the simultaneous application of individual physical, physicochemical, biological, and/or genetic strategies. Additionally, during the course of this research we continually improved our methods of zebra transgenesis and genetic construct design to achieve the objectives of this project.
Publications
- Wong, A.C., and A.L. Van Eenennaam. 2009. I-SceI meganuclease- and Tol2 transposon-mediated transgenesis methods compared in zebrafish: Transgenesis frequencies and germline transmission rates Abstract # P548. Final program and abstract guide. XVII International Plant & Animal Genome Meeting, San Diego, CA, Jan.10-14, 2009. http://www.intl-pag.org/17/abstracts/P05p_PAGXVII_548.html
- Wong, A.C., B. W. Draper and A.L. Van Eenennaam. 2009. Cre/loxP and FLP/FRT site-specific recombination systems for the biological containment of transgenic fish. Transgenic Animal Research Conference VII. August 17-22, 2009. Tahoe City, CA. Transgenic Research (in press).
- Wong, A.C. 2009. Two Germ Cell-Driven Site-Specific Recombination Systems for the Genetic Containment of Transgenic Fish. Ph.D. dissertation. University of California, Davis. Wong, A.C., B. W. Draper and A.L. Van Eenennaam. 2009. Cre and FLPe RNA function in zebrafish embryos. Transgenic Research. (pending).
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Progress 09/01/05 to 08/31/09
Outputs
OUTPUTS: To ameliorate some of the concerns associated with the release or escape of transgenic fish, a novel, non-sterility based biological containment method was devised that utilizes two site-specific recombination systems. In this system, the FLP-recombinase mediated removal of an FRT-flanked gene-of-interest triggers the Cre-recombinase mediated self-excision of the recombinase genes, leaving behind a single 34-bp loxP footprint. The recombinases in this strategy are driven by a germ cell-specific promoter such that only gametes excise the transgene, thus retaining the beneficial traits associated with the gene-of-interest in somatic tissue. To assay the efficiency of site-specific recombination in transgenic fish carrying a construct consisting of a green fluorescent protein (GFP) marker transgene flanked by both FRT and loxP sites, FLPe recombinase capped RNA was microinjected into single cell stage zebrafish embryos obtained by crossing hemizygous transgenic males with wild-type females. By 48 hours post fertilization (hpf), the proportion of embryos displaying green fluorescence following FLPe RNA microinjection was significantly lower (7.7%; p<0.001) than expected (50%) if excision had not occurred. Embryos that retained fluorescence displayed marked mosaicism. Inheritance of the excised transgene in non-fluorescent, transgenic embryos was verified by PCR amplification of a recombinase-mediated excision product, and FLPe-mediated recombination was confirmed by DNA sequencing. When sperm samples from phenotypically mosaic fish were analyzed, individuals were identified where the sperm was also positive for a Cre-excision PCR product, indicating that the loxP-flanked transgene had also been excised from a portion of the germ cells. As a proof-of-concept, this showed that two site-specific recombinases could be used to sequentially excise transgenes, including the transgenes encoding the recombinases themselves, from the germline of transgenic fish. We then tested this system in vivo by crossing separate lines of transgenic fish, each carrying a construct designed to remove transgenic DNA from the gametes of fish carrying a copy of both constructs. However, the germline transgene excision frequency observed in the double transgenic fish very low. Although the results of the flpe RNA microinjections appeared promising and offered a working proof-of-concept, ultimately the real world application of two germ cell-driven site-specific recombination systems for the genetic containment of transgenic fish was far too inefficient for this strategy to be a utilized as a means of genetic containment for transgenic fish. The results of this research project have been disseminated to the research community through conferences and publications in scientific journals, including a review paper entitled "Transgenic Approaches for the Reproductive Containment of Genetically Engineered Fish" that was published in the journal Aquaculture in 2008. Additionally several general audience publications have resulted from this project including fact sheets. Finally, results were shared with research and regulators at the 2009 Transgenic Animal Conference. PARTICIPANTS: Individuals: Dr. Alison Van Eenennaam (Principal Investigator) set the framework for this project and provided guidance to graduate students to meet specific objectives outlined in the proposal. Andrew Wong was the graduate student who worked full time on this project and who designed and created all the constructs in this project. He tested various constructs in yeast before implementing them in zebrafish. He microinjected all the constructs into zebrafish and performed all the screening and genotyping. He completed his Ph.D. dissertation on this project. Partner Organizations: Aqua Bounty Technologies allowed Andrew Wong to participate in a 3-month internship at the Aqua Bounty Technologies in San Diego. Collaborators: Dr. Bruce Draper is an Assistant Professor at UC Davis who is studying the development and maintenance of germline stem cells in zebrafish. Dr. Draper has setup a zebrafish rearing facility at UC Davis and provided expertise related to zebrafish rearing and transgenesis as a collaborator on this project. Training and professional development: This project provided training and professional development opportunities for a graduate student to undertake all of the experimental work, and understand the biology and genetics behind reproductive containment of transgenic fish, and the issues associated with their commercialization. Andrew Wong, the Ph.D. student who worked on this project, recently applied for a position with the FDA transgenic animal regulatory group. TARGET AUDIENCES: Research results were presented at the XVII International Plant & Animal Genome Meeting, Jan.10-14, 2009. San Diego, CA. The results of this project were presented to transgenic scientists and regulators charged with the regulation of genetically engineered animals at the Transgenic Animal Research Conference VII. August 17-22, 2009. Tahoe City, CA. PROJECT MODIFICATIONS: As reported last year lack of gonadal fluorescent protein expression from the male line construct led us to try a new zebrafish germ cell-specific promoter, ziwi. Additionally, as we were having difficulty identifying transgenic founder fish using traditional transgenesis methods, we adopted a transposon-mediated transgenesis technique to increase our chances of obtaining germline positive transgenic founder fish. These two modifications delayed the completion of this project for an additional year.
Impacts
The development of fast-growing, transgenic fish that efficiently use resources (time, feed, etc.) and produce less waste could result in decreased costs for the aquaculturist and a more affordable, sustainable fish supply for consumers. However, the environmental risk associated with the escape and interbreeding of genetically engineered fish with wild populations is considered to be the greatest science-based concern facing the animal biotechnology industry. There is a need for biocontainment strategies to minimize the ecological risks of transgene flow from genetically engineered fish into native fish populations. This pilot study was designed to determine the efficacy of recombinase-mediated excision events to selectively eliminate transgenic DNA from the germ cells of genetically-engineered zebrafish. Although we were able to reduce our approach to practice, the low frequency of germline transgene excision events revealed that our approach to use two germ cell-driven site-specific recombination systems was not sufficiently robust to guarantee containment genetic containment of transgenic fish should they escape into the wild and mate with native populations of fish. Other methods for biological or genetic containment of transgenic fish have also failed to achieve 100% containment. Currently it appears that, if complete containment is the goal, the most foolproof approach may involve the simultaneous application of individual physical, physicochemical, biological, and/or genetic strategies. Additionally, during the course of this research we continually improved our methods of zebra transgenesis and genetic construct design to achieve the objectives of this project.
Publications
- Wong, A.C., and A.L. Van Eenennaam. 2009. I-SceI meganuclease- and Tol2 transposon-mediated transgenesis methods compared in zebrafish: Transgenesis frequencies and germline transmission rates Abstract # P548. Final program and abstract guide. XVII International Plant & Animal Genome Meeting, San Diego, CA, Jan.10-14, 2009. http://www.intl-pag.org/17/abstracts/P05p_PAGXVII_548.html
- Wong, A.C., B. W. Draper and A.L. Van Eenennaam. 2009. Cre/loxP and FLP/FRT site-specific recombination systems for the biological containment of transgenic fish. Transgenic Animal Research Conference VII. August 17-22, 2009. Tahoe City, CA. Transgenic Research (in press).
- Wong, A.C. 2009. Two Germ Cell-Driven Site-Specific Recombination Systems for the Genetic Containment of Transgenic Fish. Ph.D. dissertation. University of California, Davis. Wong, A.C., B. W. Draper and A.L. Van Eenennaam. 2009. Cre and FLPe RNA function in zebrafish embryos. Transgenic Research. (pending).
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Since our last progress report, we have identified several lines of germline positive zebrafish that contain the female line construct. These germline founder fish were obtained via I-SceI meganuclease-mediated transgenesis as well as through Tol2 transposon-mediated transgenesis methods. Germline positive founder fish containing the female line construct produced via I-SceI meganuclease-mediated transgenesis were outcrossed to WT zebrafish and a Southern blot was performed on the F1 progeny to characterize insertions and copy number. This information was disseminated to collaborators at weekly lab meetings. Zebrafish were also microinjected with the male line construct via I-SceI meganuclease- and Tol2 transposon-mediated transgenesis methods. Sperm from F0 males was genotyped and positive males were outcrossed to WT eggs to produce F1 fish. Sperm squeezed from F1 males genotyped positive for the transgene. However, fluorescence microscopy on these hemizygous males did not reveal red gonads. For the Tol2 generated fish, reverse transcription PCR on mRNA isolated from testes from males whose sperm genotyped positive did not reveal any expression of the transgene. This lack of expression in the male line construct led us to re-evaluate the design of the construct and improvements were made. A more detailed description of what changes were made will be discussed in the Project Modifications section. The redesigned male line constructs have been microinjected and will be screened for expression when they reach the appropriate stage of development. PARTICIPANTS: Individuals Dr. Alison Van Eenennaam (Principal Investigator) set the framework for this project and provided guidance to graduate students to meet specific objectives outlined in the proposal. Andrew Wong was the graduate student who designed and created all the constructs in this project. He tested various constructs in yeast before implementing them in zebrafish. He microinjected all the constructs into zebrafish and performed all the screening and genotyping. Collaborators and contacts Dr. Bruce Draper is an Assistant Professor at UC Davis who is studying the development and maintenance of germline stem cells in zebrafish. Dr. Draper has setup a zebrafish rearing facility at UC Davis and provides expertise related to zebrafish and transgenesis. Dr. John Buchanan is the Eastern Laboratory Director of Aqua Bounty Technologies. Dr. Buchanan was the contact at Aqua Bounty Technologies who agreed to allow Andrew Wong to participate in a 3-month internship at Aqua Bounty Technologies in San Diego. Dr. Xavier Lauth is a Senior Research Scientist at Aqua Bounty Technologies. Dr. Lauth worked closely with Andrew Wong to help produce germline positive transgenic fish utilizing the I-SceI meganuclease-meditated transgenesis technique. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Lack of expression from the male line construct led us to try a new zebrafish germ cell-specific promoter, ziwi. The ziwi promoter is more advantageous for our containment strategy because it is expressed sooner and in more cell stages than the vasa promoter. We employed Gateway cloning technology to create the new construct and took this opportunity to address any weaknesses in the original design of both the male and female line constructs. For the male line construct, we were concerned that the FLPe::DsRed translation fusion protein may be interfering with recombinase or fluorescence expression or both so we elected to replace that portion with a FLPe-IRES-EGFP segment. The internal ribosome entry site (IRES) would link the expression of the recombinase and the fluorescent marker protein without physically joining them together. EGFP replaced DsRed because EGFP is brighter and easier to screen for than the dimmer DsRed. Similar improvements were made to the female line construct; the ziwi promoter replaced the vasa promoter and Cre::ECFP translational fusion protein was replaced with Cre only to simplify the construct.
Impacts
The lack of expression from the male line construct led us to a change in knowledge and a change in actions. In an effort to increase the chances of obtaining a germline positive founder fish containing the male line construct, we employed a new zebrafish germ cell-specific promoter, ziwi, and a new way to build constructs using the Gateway cloning technology. A collaborator, Dr. Bruce Draper, was studying ziwi, a zebrafish homolog of the Drosophila piwi, involved in proliferation and maintenance of germline stem cells. His research revealed that the ziwi promoter turned on sooner and was active in more cell stages than the vasa promoter we were using. He suggested we use promoter in our male and female line constructs as it would be advantageous to have the male and female line constructs turn on as soon as possible in the germ cells. To expedite the vector construction process, we adapted our constructs to use Gateway cloning techniques instead of relying on slower traditional sub-cloning methods. The Gateway process was specifically tailored for use with zebrafish and incorporated the Tol2 transgenesis method.
Publications
- No publications reported this period
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Progress 09/01/06 to 08/31/07
Outputs
OUTPUTS: Since our last progress report, we began microinjection of linearized versions of the completed male line vectors. Using traditional microinjection techniques, we microinjected thousands of one-cell stage zebrafish embryos, however, we were unable to identify germ line founders for the male line vectors. Following fluorescent screening of in vitro fertilized eggs, we were unable to identify any offspring with red gonads. To increase the efficiency of producing germ line positive zebrafish, we placed our male line constructs into a Tol2 transposon vector and co-microinjected them with Tol2 mRNA into one-cell stage zebrafish embryos. These fish are now sexually mature and we are in the process of screening them for the transgene. We will use traditional fluorescent microscopy as well as sperm and egg genotyping to identify germ line positive fish. We completed the female line vector and microinjected the linearized DNA into one-cell stage zebrafish embryos. Using fluorescent
microscopy, we indentified zebrafish that were GFP positive, indicating that they were expressing the female line vector. We are in the process of outcrossing those individuals to wild-type zebrafish to identify transgenic founders that are germ line positive for the female line vector. In addition to Tol2 transposon-mediated transgenesis, we also recently began I-SceI meganuclease-mediated transgenesis to further improve our chances of producing germ line positive zebrafish. Both male line vectors and the female line vector were adapted to work with this new transgenesis method. Circularized vectors were incubated with I-SceI meganuclease for one hour before microinjection into one-cell stage zebrafish embryos. The incubation time allows for the meganuclease to excise the entire transgene construct from the vector, linearizing it. The meganuclease remains attached to both ends of the transgene construct and it improves the early, stable integration of the transgene construct into the
genome. Zebrafish embryos microinjected with this technique are less than a month old. We have already observed uniform GFP expression in some zebrafish fry expressing the female line vector with this new microinjection technique. When these fish reach sexually maturity, we will outcross and genotype them to identify germ line positive founders. Given the extensive time required to screen and raise germ line positive founder zebrafish for each construct, we requested a one year no-cost extension. This also enabled us to utilize new microinjection techniques such as the Tol2 transposon-mediated and I-SceI meganuclease-mediated transgenesis methods. We will use the one year no-cost extension to identify and raise germ line positive founder zebrafish to maturity. We will then cross the two lines of fish and monitor fluorescence in the muscle tissue and gonads of the F1 progeny. The genotype of these fish will be assessed from finclips as well as from sperm and eggs. Following
confirmation that sperm and egg from F1 fish lack the transgene, F1 fish will be outcrossed to wild-type zebrafish. F2 progeny should appear completely wild-type and lack the appearance of any type of fluorescence.
PARTICIPANTS: Andrew Wong, PhD student
TARGET AUDIENCES: Scientific community with an interest in Aquaculture
PROJECT MODIFICATIONS: We requested a no-cost one-year extension to this grant as it has taken longer than anticipated to get transgenic zebrafish founder lines.
Impacts
The development of fast-growing, transgenic fish that efficiently use resources (time, feed, etc.) and produce less waste could result in decreased costs for the aquaculturist and a more affordable, sustainable fish supply for consumers. However, the environmental risk associated with the escape and interbreeding of genetically engineered fish with wild populations is considered to be the greatest science-based concern facing the animal biotechnology industry. Computer simulations have suggested that in specific circumstances the escape of fast-growing transgenic fish could lead to the eventual extinction of native fish populations. There is a need for biocontainment methodologies to minimize the ecological risks of transgene flow from genetically engineered fish into native fish populations. This study is a pilot study to determine the efficacy of recombinase-mediated excision events to selectively eliminate transgenic DNA from the germ cells of genetically-engineered
zebrafish. This information will provide the data needed to determine whether this is a feasible approach for the biocontainment of genetically engineered fish should they escape into the wild and mate with native populations of fish. The success of this project could provide the aquacultural industry with a technique to limit the spread of transgenic DNA from commercialized lines of genetically-engineered fish thereby protecting native fish populations from the risk of transgene spread, while helping to realize the potential economic benefits that could be derived from the genetic manipulation in fish.
Publications
- A. C. Wong, and A. L. Van Eenennaam. 2008. Transgenic Approaches for the Reproductive Containment of Genetically Engineered Fish. Aquaculture. In press..
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Progress 09/01/05 to 09/01/06
Outputs
We are evaluating the use of site-specific recombination systems to excise transgenic DNA from the gametes of zebrafish (Danio rerio) as an approach towards biological containment of genetically engineered fish. Our experimental design relies on the use of recombinase/fluorescent marker fusion proteins to track the excision of the target transgene. We have been working on confirmation of the dual functionality (i.e. recombinase and fluorescence) of these fusion proteins as outlined in the experimental plan. Our first objective was to confirm the dual functionality of recombinase/fluorescent marker fusion proteins in Schizosaccharomyces pombe. We have completed Objective 1A, the design and construction of Cre/ECFP and FLPe/DsRed-Express translational fusion proteins, and Objective 1B, the confirmation of dual functionality of the recombinase/fluorescent translational fusion proteins in S. pombe. For Objective 1A, two constructs were created, one containing a Cre/ECFP
translational fusion protein and the other containing an FLPe/DsRed-Express translational fusion protein. Each construct was inserted into a separate yeast shuttle vector. For Objective 1B, the fluorescence activity of the two recombinase/fluorescent fusion proteins was confirmed through visual observations with a fluorescent microscope. Two additional constructs, a loxP tester plasmid and an FRT tester plasmid, were constructed to test the recombinase activity of the fusion proteins. The loxP tester plasmid included a GFP ORF flanked on either side by loxP sites, whereas the FRT tester plasmid included a GFP ORF flanked on either side by FRT sites. Transformation of yeast carrying the appropriate tester plasmid with either the Cre/ECFP or the FLPe/DsRed-Express translational fusion protein resulted in the excision of the GFP ORF from the tester plasmid, confirming the recombinase functionality of the fusion proteins. Correct excision junctions were confirmed by PCR and DNA
sequencing. Retention of dual functionality of the recombinase/fluorescent fusion proteins was a critical milestone as fluorescence activity is going to be used for visually tracking the presence of the transgenic DNA in zebrafish. Following the completion of Objective 1, we commenced work on Objective 2A, the development of male and female line vectors for in vivo studies in zebrafish. We have completed the male line construct containing the FLPe/DsRed fusion driven by a zebrafish vasa promoter. We began construction of the female line construct which features a GFP reporter gene driven by a zebrafish muscle-specific promoter sandwiched between a vasa promoter and a Cre/ECFP fusion and flanked by loxP sites, but found that the Cre/ECFP was recombining the loxP sites within the female line construct while we were assembling it in Escherichia coli. Expression of the Cre/ECFP protein was not expected as it not located downstream of a promoter. To solve this problem we made use of the
fact that prokaryotes are unable to process introns and inserted a 150 bp zebrafish intron into the Cre gene to prevent the expression of active Cre in E. coli. We are now completing the construction of the female line construct.
Impacts
The development of fast-growing, transgenic fish that efficiently use resources (time, feed, etc.) and produce less waste could result in decreased costs for the aquaculturist and a more affordable, sustainable fish supply for consumers. However, the environmental risk associated with the escape and interbreeding of genetically engineered fish with wild populations is considered to be the greatest science-based concern facing the animal biotechnology industry. Computer simulations have suggested that in specific circumstances the escape of fast-growing transgenic fish could lead to the eventual extinction of native fish populations. There is a need for biocontainment methodologies to minimize the ecological risks of transgene flow from genetically engineered fish into native fish populations. This study is a pilot study to determine the efficacy of recombinase-mediated excision events to selectively eliminate transgenic DNA from the germ cells of
genetically-engineered zebrafish. This information will provide the data needed to determine whether this is a feasible approach for the biocontainment of genetically engineered fish should they escape into the wild and mate with native populations of fish. The success of this project could provide the aquacultural industry with a technique to limit the spread of transgenic DNA from commercialized lines of genetically-engineered fish thereby protecting native fish populations from the risk of transgene spread, while helping to realize the potential economic benefits that could be derived from the genetic manipulation in fish.
Publications
- No publications reported this period
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Progress 09/01/05 to 08/31/06
Outputs
We are evaluating the use of site-specific recombination systems to excise transgenic DNA from the gametes of zebrafish (Danio rerio) as an approach towards biological containment of genetically engineered fish. Our experimental design relies on the use of recombinase/fluorescent marker fusion proteins to track the excision of the target transgene. We have been working on confirmation of the dual functionality (i.e. recombinase and fluorescence) of these fusion proteins as outlined in the experimental plan. Our first objective was to confirm the dual functionality of recombinase/fluorescent marker fusion proteins in Schizosaccharomyces pombe. We have completed Objective 1A, the design and construction of Cre/ECFP and FLPe/DsRed-Express translational fusion proteins, and Objective 1B, the confirmation of dual functionality of the recombinase/fluorescent translational fusion proteins in S. pombe. For Objective 1A, two constructs were created, one containing a Cre/ECFP
translational fusion protein and the other containing an FLPe/DsRed-Express translational fusion protein. Each construct was inserted into a separate yeast shuttle vector. For Objective 1B, the fluorescence activity of the two recombinase/fluorescent fusion proteins was confirmed through visual observations with a fluorescent microscope. Two additional constructs, a loxP tester plasmid and an FRT tester plasmid, were constructed to test the recombinase activity of the fusion proteins. The loxP tester plasmid included a GFP ORF flanked on either side by loxP sites, whereas the FRT tester plasmid included a GFP ORF flanked on either side by FRT sites. Transformation of yeast carrying the appropriate tester plasmid with either the Cre/ECFP or the FLPe/DsRed-Express translational fusion protein resulted in the excision of the GFP ORF from the tester plasmid, confirming the recombinase functionality of the fusion proteins. Correct excision junctions were confirmed by PCR and DNA
sequencing. Retention of dual functionality of the recombinase/fluorescent fusion proteins was a critical milestone as fluorescence activity is going to be used for visually tracking the presence of the transgenic DNA in zebrafish. Following the completion of Objective 1, we commenced work on Objective 2A, the development of male and female line vectors for in vivo studies in zebrafish. We have completed the male line construct containing the FLPe/DsRed fusion driven by a zebrafish vasa promoter. We began construction of the female line construct which features a GFP reporter gene driven by a zebrafish muscle-specific promoter sandwiched between a vasa promoter and a Cre/ECFP fusion and flanked by loxP sites, but found that the Cre/ECFP was recombining the loxP sites within the female line construct while we were assembling it in Escherichia coli. Expression of the Cre/ECFP protein was not expected as it not located downstream of a promoter. To solve this problem we made use of the
fact that prokaryotes are unable to process introns and inserted a 150 bp zebrafish intron into the Cre gene to prevent the expression of active Cre in E. coli. We are now completing the construction of the female line construct.
Impacts
The development of fast-growing, transgenic fish that efficiently use resources (time, feed, etc.) and produce less waste could result in decreased costs for the aquaculturist and a more affordable, sustainable fish supply for consumers. However, the environmental risk associated with the escape and interbreeding of genetically engineered fish with wild populations is considered to be the greatest science-based concern facing the animal biotechnology industry. Computer simulations have suggested that in specific circumstances the escape of fast-growing transgenic fish could lead to the eventual extinction of native fish populations. There is a need for biocontainment methodologies to minimize the ecological risks of transgene flow from genetically engineered fish into native fish populations. This study is a pilot study to determine the efficacy of recombinase-mediated excision events to selectively eliminate transgenic DNA from the germ cells of
genetically-engineered zebrafish. This information will provide the data needed to determine whether this is a feasible approach for the biocontainment of genetically engineered fish should they escape into the wild and mate with native populations of fish. The success of this project could provide the aquacultural industry with a technique to limit the spread of transgenic DNA from commercialized lines of genetically-engineered fish thereby protecting native fish populations from the risk of transgene spread, while helping to realize the potential economic benefits that could be derived from the genetic manipulation in fish.
Publications
- A. L. Van Eenennaam and P. G. Olin. 2006. Careful risk assessment needed to evaluate transgenic fish. California Agriculture 60(3) 126-131.
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