Progress 09/15/06 to 02/28/11
Outputs
OUTPUTS: In the previous year, we showed that FLP-FRT system is useful for site-specific gene integration when FLPe is used for catalyzing recombination. This helped us in solving the major bottleneck in the proposed technology i.e. development of an alternative recombination system (other than Cre-lox) for site-specific gene integration. To accomplish the overall goal of developing marker-free site-specific gene integration technology, we developed 4 rice (Nipponbare) "target lines" that contain FRT target site and heat-inducible cre gene and lox sites. The cre-lox components are designed to remove marker genes subsequent to FLP-mediated gene integration. We also developed new "donor vector" in which both lox sites and FRT sites are incorporated. This donor vector is designed for gene integration based on FRT x FRT recombination and marker removal based on loxP x loxP recombination. By particle bombardment of donor vector, we developed 8 site-specific integration lines derived from 2 target lines. Some of these lines contain a only site-specific integration into target locus (single copy) and others contain both site-specific and random integrations (multi-copy). Marker excision experiments involving heat-induction of cre gene will be carried out on these lines. The major observations are: (1) heat-inducible cre gene remains silent through tissue culture phase as a result a stable site-specific integration locus is obtained at high efficiency, (2) FLP-FRT mediated site-specific integration locus is stable and precise. PARTICIPANTS: Soumen Nandy, postdoc Jamie Underwood, Research Technician TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The use of two separate recombination systems in a single platform was will allow the development of a Marker-free site-specific gene integration technology. This technology will allow formation of a precise transgene locus that is devoid of undesirable marker genes. Transgene integration via enzyme (recombinase) mediated recombination reaction will minimize or eliminate any 'unintended' genetic aberrations produced by poking of T-DNA or particle injury in the genome. The transgene locus produced in this manner will be easy to characterize as its position will be fixed in the genome. Finally, this technology could be improved to allow gene stacking i.e. incorporation of multiple gene array into a selected genomic site.
Publications
- Khattri A, Nandy S, and Srivastava V (2011) Heat-inducible Cre-lox system for marker excision in transgenic rice. J. Biosciences DOI 10.1007/s12038-011-9010-8.
- Srivastava V, Akbudak MA, Nandy, S (2011) Marker-free plant transformation. In Plant Transformation Technology Revolution in Last Three Decades. Bentham Science Publishers (Eds: Y. Dan and D. W. Ow). (in press).
- Srivastava V and Ow D (2010) Site-specific recombination for precise and clean transgene integration in plant genome (Chapter 13). In Plant Transformation Technologies, Blackwell publishing (Eds: C. Neal Stewart, Jr., Alisher Touraev, Vitaly Citovsky, and Tzvi Tzfira).
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Progress 09/15/09 to 09/14/10
Outputs
OUTPUTS: The goal of this project is to develop marker-free site-specific gene integration method. For this purpose, two different recombinase systems were employed: Cre-lox and FLP-FRT. Based on the previous year's data, Cre-lox was dedicated for marker gene excision, while FLP-FRT was used for transgene integration. Following major observations were made: (i) Efficiency of FLP-FRT mediated site-specific gene integration in rice is 10 - 30%, and therefore suitable for rice transformation. (ii) Co-bombardment of improved FLP gene, FLPe, is necessary for obtaining site-specific integration of the introduced transgene. (iii) Site-specific integration locus contains the expected precise structure and expresses the transgene (GUS) stably through subsequent generations. Next, the founder lines were transformed with a new transgene construct that contains two loxP sites marker removal. The resulting integration locus will contain 4 loxP sites to allow removal of flanking genomic sequences leaving only the gene-of-interest behind (marker-free locus). Twelve lines were developed, 3 of which were single-copy. These lines were heat-shocked to induce the Cre gene and initiate loxP x loxP recombination. This recombination is expected to generate marker free locus. Transgenic plants from the single-copy lines have been transferred to greenhouse. Heat-treatment on the callus lines was done and genomic DNA tested for the presence of the recombination footprint. Each line was verified to contain the footprint upon heat treatment. These data validate proper functionality of each component: FLPe-FRT system for stable gene integration, and heat-inducible Cre for marker excision. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Soumen Nandy, postdoc, worked on making DNA constructs, rice transformation and transgenic line analysis. M. Aydin Akbudak, graduate student, worked on the assessing the efficiency of different FLP versions. Vibha Srivastava, PI, directed the project and analyzed data. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This research developed FLPe-FRT mediated gene integration system for rice. The method is likely to work on other crops also. Further, use of two separate recombination systems in a single platform was successful. This demonstrates that each system works properly to carry out the dedicated task. These tools are critical for developing Marker-free site-specific gene integration technology. The project involved one graduate student and one postdoc.
Publications
- (1) Nandy S, Srivastava V (2010) Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system. Plant Biotechnol J. doi: 10.1111/j.1467-7652.2010.00577.x.
- (2) Akbubak MA, More, A, Nandy S, Srivastava V (2010) Dosage-dependent gene expression from direct repeat locus in rice developed by site-specific gene integration. Mol. Biotechnol. 45(1):15-23.
- (3) Srivastava V and Gidoni D (2010) Site-specific gene integration technologies for crop improvement. In Vitro Cellular and Developmental Biology - Plant DOI: 10.1007/s11627-009-9274-y
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Progress 09/15/08 to 09/14/09
Outputs
OUTPUTS: Two approaches were taken to establish a marker-free site-specific integration system: (1) Use Cre-lox for integration and heat-shock FLP for subsequent excision of marker gene, (2) Use FLP-FRT for integration and heat-shock Cre for subsequent excision of marker gene. In approach (1) following conclusions were derived: (i) Cre-lox mediated site-specific integration of foreign gene occurred efficiently but heat-induced FLP activity was not sufficient to induce excision of FRT flanked maker genes; (ii) even stronger FLP activity supplied via stable integration of a FLP gene did not induce FRT x FRT recombination; (iii) introduction of FLPe activity (a modified FLP gene) was finally successful in inducing recombination; (iv) FLPe mediated recombination resulted in both excision of marker genes and inversion of the gene-of-interest. This conclusion is based on Southern analysis and sequencing of PCR products. In approach (2) following progress was made: (i) Single-copy lines containing the target structure were generated and verified by Southern analysis; (ii) FLP-FRT mediated site-specific integration failed, which was attributed to downregulation of FLP activity by the presence of FRT site between promoter and gene; (ii) co-bombardment of FLPe or FLPo genes resulted in successful site-specific integration of the gene-of-interest. PARTICIPANTS: Soumen Nandy, postdoc, worked on rice transformation, molecular analysis. M. Aydin Akbudak, graduate student, worked on comparing FLP, FLPe, FLPo activities and their use for excision. Vibha Srivastava, PI, designed and supervised the project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This research identified problems associated with FLP-FRT system for genome manipulation of plants. In addition, it devised solutions to optimize integration and excision process. In the final year, molecular validation will be done to determine precision and efficiency of the process. Involvement of a graduate student and a postdoc allowed us to test two parallel strategies, although, they were not initially planned but had to be included as a contingency plan.
Publications
- Akbudak MA, Srivastava V. 2009. Marker-free site-specific gene integration for plants. XVII Plant and Animal Genome conference, San Diego, Jan 2009.
- Nandy S, Srivastava V. 2009. Intermolecular recombination efficiencies of FLP-FRT and Cre-lox in rice. IPMB Congress, St. Louis, Oct 2009.
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Progress 09/15/07 to 09/14/08
Outputs
OUTPUTS: The main objective of this project is to test a molecular strategy of marker-free site-specific integration using one of the two recombination systems (Cre-lox and FLP-FRT) for site-specific integration and the other for marker excision. To decide which of the two systems should be employed for site-specific integration, pros and cons of both systems were considered but no clear decision could be made. The dilemma was specifically based on the fact that Cre-lox is a proven integration system and FLP-FRT is not and Cre-lox is more efficient for marker excision compared to FLP-FRT. Therefore, two possible approaches were undertaken: (a) use FLP-FRT for integration and Cre-lox for excision, (b) use Cre-lox for integration and FLP-FRT for excision. PARTICIPANTS: 1. M. Aydin Akbudak, Graduate student, worked on rice transformation with pAK7 and pRP4, isolated tranegnic lines and characterized recombinase expression and locus structure. Aydin also carried out heat-induction experiments to assess FLP-mediated excision. He will continue to work on this project. Aydin's stipend was paid from the grant. 2. Soumen Nandy, postdoc associate, made pNS5 and pAM18 constructs, generated transgenic lines using pNS5 vector. Carried out Southern analysis. He also perfomed assays to test relative of efficiencies of Cre-lox and FLP-FRT sysems for a bi-molecular recombination. Soumen's salary is paid from the grant. 3. Vibha Srivastava, PI, no salary drawn from the grant. Vibha developed design and plan of the project, directed and trained both individuals and interpreted data. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Following major changes occurred: (1) Delay in hiring postdoc by almost 10 months, slowed the project in its first year. (2) The project was expanded to test two approaches instread of only one: the proposal described testing the strategy with FLP-mediated integration and Cre-mediated excision. In addition to this approach, Cre-mediated integration and FLP mediated excision is also included in this project. (3) An unexpected outcome is that only 5 out of 42 pNS5 (FLP-FRT construct) lines are single-copy, which indicates that FLP activity is rather weak in recombining out extra copies of pNS5 to generate single-copy, as is normally observed in Cre-lox constructs. If that would turn out to be true, we have to abandon FLP-FRT system for another recombination system such as R-RS or phiC31. (4) Another expected outcome is that heat-inducible FLP was highly inefficient in marker excision. Therefore, new versions of FLP, FLPe and FLPo will be tested. These expected reults expanded the project and significantly increased the amount of additional work needed to get most out of this project.
Impacts
Development of DNA constructs: Two separate sets of constructs were developed: (a) pAK7 and pRP4 to facilitate Cre-lox mediated integration and FLP-FRT mediated excision, (b) pNS5 and pAM18 to facilitate FLP-FRT mediated integration and Cre-lox mediated excision. pAK7 contains a lox76, which serves as a target site, it also contains a heat-shock promoter (HSP) driven FLP gene. The whole construct is flanked by oppositely oriented FRT sites. Similarly, pNS5 contains FRTL, which serves as the target site, and it also contains HSP-Cre. The whole pNS5 construct is flanked by appositely oriented loxP sites. Both pAK7 and pNS5 are binary vectors, and therefore transferred to Agrobacterium tumefaciens strain EHA105. Rice variety Nipponbare was transformed with these vectors to develop two pAK7 lines, and 42 pNS5 lines. Each of these lines was characterized by Southern hybridization, RT-PCR for cre and FLP expression and presence of various lox and FRT sites. Both pAK5 lines were full-length single-copy, expressed cre constitutively but FLP only upon 2h of 42 C treatment. Room temp FLP activity was negligible. Of the 42 pNS5 lines, only 5 were full-length single-copy. The expression data on these lines is awaited. Particle bombardment of pAK5 lines with pRP4 generated 2 integration lines: 1.1 and 1.2. Both of these integration lines were structurally intact and precise as determined by Southern blot and sequencing of PCR amplified segments. Both lines were subjected to heat treatment based on a protocol developed earlier. Heat (42 C) was applied recurrently for a few days and DNA extracted to analyze FLP-mediated marker excision by PCR and Southern. While Southern analysis did not reveal any excision, PCR of one of the several treated samples indicated excision of the right flanking sequence (flanked by FRT sites). Overall, heat-inducible FLP-FRT system, appears to be inefficient for excisions from genomic sites, though cre-lox was highly efficient in generating precise integration structures. In separate experiment, competency of FLP-FRT and Cre-lox systems was compared in a bi-molecular recombination reaction. This experiment was done to assess if FLP-FRT is a useful system for carrying out site-specific integration in plant cells. This experiment revealed that an extra-chromosomal bi-molecular recombination using FLP-FRT system is highly inefficient. While a number of Cre-lox integrant lines were recovered, not a single FLP-FRT lines were obtained.
Publications
- 1. Srivastava V (2008) Site-specific recombination mediated genetic engineering of rice. The abstract book of the international conference of Plant & Animal genome XVI, San Diego CA, Jan 12-16, 2008. p91.
- 2. Srivastava V (2008) Plant Genome Manipulation using Cre/lox Technology. Annual conference of the Society of In Vitro Biology, Vol 44, pS21.
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Progress 09/15/06 to 09/14/07
Outputs
The goal of this project is to test the proposed molecular strategy for combining two separate applications of recombinase technology: recombinase-mediated site-specific gene integration and recombinase-mediated marker excision. The combined "marker-free site-specific gene integration technology" technology will allow to draw benefits of each of these applications i.e. stable gene expression as a result of site-specific gene integration, and environmentally improved product as a result of marker gene removal. The molecular strategy is based on two different recombination systems, FLP/FRT and Cre/lox. The strategy also mandates that marker excision system should be tightly regulated. We chose soybean heat shock (HS) promoter (HSP17.5E) for regulating recombinase gene expression. Therefore, in the first year of the project, we compared the efficiency of HS-Cre and HS-FLP. For this purpose, we generated transgenic rice callus containing either of these genes along with a
recombination test construct (as described in the project). A recombination reaction is expected to generate GUS expression by looping-out a fragment between promoter and the GUS gene. We treated the callus with 42 oC for 3 hours and incubated at room temp for 72 hours prior to assessing GUS activity. We found that HS-Cre was several fold more efficient than HS-FLP. We are in the process of testing a HS-Cre transgenic plant to test HS-Cre activity in leaf tissue. This data indicates that HS-Cre should be used for marker excision step, which is a non-selectable step and therefore should occur at high efficiency. The above data also indicates that FLP/FRT should be utilized to site-specific gene integration step. As no report of FLP-mediated gene integration is available, we are first testing the efficiency and efficacy of FLP/FRT-mediated gene integration. For this purpose, we developed a plasmid construct containing Ubi_promoter-FRTL-FLP gene construct (called pSM34 in our lab). Maize
ubiquitin-1 promoter is expected to generate strong FLP expression. FRTL that contains a single base mutation in its left binding element is the target site for site-specific gene integration step. We have developed two rice transgenic lines (34.6 and 34.7) with pSM34 that contain intact full-length integration of the construct. Currently, we are doing FLP expression analysis of these lines using RT-PCR and Northern blot. We have also developed a donor plasmid that will be utilized for inserting GUS gene into FRTL target site in 34.6 and 34.7 lines. Finally, we have started constructing the target plasmid that will be used for testing marker-free site-specific gene integration strategy. At this time, we have generated individual components of this tartget construct: Ubi-FRTL-FLP gene, HS-Cre gene and 35S-HPT gene for site-specific gene integration, marker excision, and transformation selection, respectively.
Impacts
1. Generate information on utility of FLP/FRT system for gene integration application. 2. Generate information on utility of HS-Cre for marker excision in callus, leaves, and germ cells. 3. Development of a new transgenic technology.
Publications
- No publications reported this period
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