Progress 09/01/10 to 08/31/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?(a) This project trained a postdoctoral associate, Dr. Soumen Nandy, who is currently seeking independent teaching and research positions. (b) the project also trained an undergraduate student, Eliot Preutt, who is studying Crop Science and Biotechnology. (c) the above trainees were provided travel support to attend annual meetings of the Society of In Vitro Biology, where they presented poster and talk. How have the results been disseminated to communities of interest?The outcomes of this project were published in journals, book chapters and presented in conferences as posters and talks. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project accomplished the goal of developing plant transformation methods for generation of marker-free transgenic plants. Using rice as the model plant and site-specific recombination systems for DNA manipulations, a marker-free site-specific integration method was developed that allowed targeted integration of transgenes into pre-determined genomic site followed by removal of all marker genes. To accomplish this goal, several technical bottlenecks had to be resolved, which were the basis of the specific objectives of this project: (1) first, the method of site-specific gene integration using FLP-FRT recombination was developed: (a) It was determined that promoter-trapping enhances the rate of recovery of the site-specific integration clones, but the expression of FLP gene is inhibited if an FRT site is located downstream of the promoter, (b) it was found that the use of thermotolerant FLPe variant was critical in recovering the clones, (b) the gene expression from the locus, as expected, was stable, and the sequence was predictable. All these indicated that FLP-FRT based site-specific integration is very precise in plants, especially, rice. (2) Next, the goal was that this locus should be converted to marker-free. For conversion to a marker-free locus Cre-lox was used. Cre-lox is most reliable, widely used recombination system, and marker-excision is non-selectable, multi-cellular reaction. Therefore, Cre-lox, instead of FLP-FRT, was dedicated to this task. Further to streamline marker-excision process, and recover marker-free clones in primary transgenic lines, inducible Cre-lox system was used: (a) it was determined that heat-inducible promoter, Gmhsp17.5E, is very efficient in heat-inductions and driving Cre-mediated marker excisions, (b) hea-inducible marker excisions were heritable by progeny, generating marker-free T1 lines, (c) Cre-lox system was extremely effcient and no further optimization was necessary as heat-inducible marker excision occurred at >99% effciency in 12 out of 20 plants, and ~50% effciency in 5 plants. Only 3 out of 20 plants failed to undergo marker excision. It should be noted that these plants could be treated again to obtain partial marker excision, (c) the transgene located in the locus showed more or less the same expression level before and after marker excision, and increased ~2x in the homozygous progeny. In summary, this project was highly successful in accomplishing its specific objectives and the overall goal. The method developed in this project laid the foundation for a gene stacking approach, which the PI is currently working on. The gene stacking approach will allow integration of multiple genes into the specified locus to resolve the complexity associated with the breeding of multiple segregating transgenes.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Srivastava V and Thomson J (2015) Gene Stacking by Recombinases. Plant Biotech J. (in press)
- Type:
Book Chapters
Status:
Published
Year Published:
2015
Citation:
Srivastava V and Ow DW (2015) Simplifying transgene locus structure through Cre-lox recombination. Plant Gene Silencing, Methods in Molecular Biology (eds. K. S. Mysore and M. Senthil-Kumar).
- Type:
Journal Articles
Status:
Submitted
Year Published:
2015
Citation:
Nandy S, Zhao S, Pathak B, Muthusamy M, and Srivastava V (___) Gene stacking in plants using recombinases for gene integration and nucleases for marker gene deletion. BMC Biotechnol (submitted)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
SRIVASTAVA V, Nandy S, and Zhao S. Iterative modification of transgene locus towards multigene stacking into a single genomic site. Society of In Vitro Biology Vol 51, Issue Abstract, Tucson AZ, May 30 - jun 3, 2015.
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Progress 09/01/13 to 08/31/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems: The initial part of the project went much more smoothly than expected, therefore, we added two more experiments to help us accomplish the overall goal of the project, which is to develop a gene stacking approach: (1) testing whether new genes can be added to the marker-free site-specific integration locus by FLP-FRT and Cre-lox system, (2) whether FLP-FRT, Cre-lox and heat-treatment have off-target genomic effects. What opportunities for training and professional development has the project provided? Dr. Soumen Nandy, postdoc, presented poster at (1) the SIVB meeting at Savannah, GA, May 31 - June 4, 2014; (2) International Workshop on Engineered Crops, Des Moines, Iowa, April 28–29, 2014 How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? In the final year of the project, targeting MF-SSI locus (B1 and B6 lines) through retransformation for the insertion of the second gene will be done, and investigation of the off-target effects that might occur as the result of the transformation process in the rice genome will be done by RNAseq analysis. The data developed in this project will be used for developing gene stacking approach for transgenic crops.
Impacts
What was accomplished under these goals?
The following questions were addressed: (1) is marker-free locus generated on parent lines C and D transmitted to the progeny, (2) how does the procedure (biolistic and heat treament) affect the plant transcriptome. In the previous year, two site-specific lines from each parent C and D were generated (C1, C2, D1, and D2). Except for C1, all plants generated marker-free site-specific integration locus upon heat treatment as verified by PCR. This year, progeny of C2, D1 and D2 were analyzed to determine inheritance of the marker-free locus. These plants were heat-treated at the seedling stage, tested for marker excision 4 weeks later, and allowed to grow till maturity in the greenhouse. C2 and D1 progeny inherited marker-free locus at near 100% efficiency i.e. among 20 progeny seedlings analyzed, either marker-free site-specific integration locus or parent locus was found as the plants were hemizygous. Transmission of marker-free locus in D2 was lower as only 3 out of 20 contained original site-specific integration locus, and 9 contained marker-free site-specific integration locus. This investigation indicates that marker-free site-specific integration locus can be developed at reasonable effcieincy by FLPe-mediated site-specific integration and heat-inducible-Cre mediated marker-excision. Towards the second question, RNAseq approach was adopted to analyze genome-wide transcriptomic changes induced by the procedures used for generating marker-free site-specific integration lines (agrobacterium, biolistic, and heat-treatment). For this, transgenic lines B were chosen. RNA was isolated from two samples each of Nipponbare (wild-type control), line B (developed by agrobacterium-mediated transformation), line B1 and B6 (biolistic delivered FLP-FRT-mediated site-specific integration of GUS gene), marker-free B1 and B6 lines (heat-treated induction of Cre-lox system to excise marker genes). A total of 12 RNA samples were sequenced on illumina platform, and the data was analyzed for identify differentially expressed genes. Bioinformatics (sequence alignments and log2 expression analysis) has been completed. Validation by qRT-PCR will be done, and final report developed in the next year.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2014
Citation:
Nguyen LD, Underwood JL, Nandy S, Akbudak MA, and Srivastava V (2014) Strong activity of FLPe recombinase in rice plant does not correlate with the transmission of the recombined locus to the progeny. Plant Biotech. Rep. (accepted)
- Type:
Book Chapters
Status:
Under Review
Year Published:
2015
Citation:
Srivastava V, Ow DW ___Simplifying transgene locus structure through Cre-lox recombination. Plant Gene Silencing, Methods in Mol. Biol.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Nandy S, Srivastava V [Poster] Marker-gene excision by Cre/lox and its stable inheritance in site-specific gene integration lines of transgenic rice generated by FLP/FRT recombination. Annual Conference of the Society of In Vitro Biology, Savannah, GA, May 31 - June 3, 2014
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Nandy S, Akbudak MA, Srivastava V [Invited Talk]
Applications of site-specific recombination systems for plant transformation. Plant Transformation Technologies II, February 12th 14nd 2014, Vienna, Austria.
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Progress 09/01/12 to 08/31/13
Outputs
Target Audience: The audience interested in the outcome of this project could include academic research communities and biotech industry. The results of this project were either published in peer reviewed journals or presented in the scientific conferences. Changes/Problems: As mentioned above, this project found that heat-inducible Cre-lox system is very efficient (>90%) in excising marker genes from the site-specific integration (SSI) locus. In the proposed project, we had planned to optimize marker-excision step through negative selection of marker-retaining SSI cells (Obj. 3). We proposed using dhlA gene as the negative selection marker. However, observed high efficiency of marker excision in lines A and B (also in T0 plants of C and D) renders Obj 3 unnecessary. Therefore, we are replacing it with a new experiment that is potentially of high interest to BRAG program: we will compare transcriptomes of lines A , B, their SSI derivatives, and the marker-free SSI derivatives. We plan on doing RNAseq. This analysis will reveal the transcriptomic changes due to tissue culture and transformations, and upon heat shock treatment and marker-excision. A very limited information on the genomic/transcriptomic changes due to tissue culture/transformation is available in literature. What opportunities for training and professional development has the project provided? Drs. Soumen Nandy and Drs. Aydin Akbudak made oral presentations in the annual meeting of SIVB at Seattle, WA, June 12-16, 2012. Mr. Linh Nguyen, a MS student, also attended this meeting and presented a poster. Dr. Nandy also attended SS-ASPB meeting in Little Rock, April 6-8, 2013, and participated in poster session. How have the results been disseminated to communities of interest? Poster presentations at SIVB and PAG conferences, invited talk at SIVB, and publications in journals and books are the method for disseminating findings of this project to the research community. What do you plan to do during the next reporting period to accomplish the goals? We plan on doing the following: (1) complete progeny analysis of SSI lines derived from C and D; (2) analyze any transgene (GUS) expression changes that may have occurred upon marker excision (marker-free SSI lines derived from A and B did not experience GUS expression changes); (3) Heat-induced Cre-lox system has so far been found to be very efficient in marker excision, therefore Obj 3 (use of dhlA gene) has been replaced with the following two experiments: (a) study transcriptomic changes induced by tissue culture and transformation in SSI lines and their marker-free SSI progeny; (2) develop a gene stacking protocol based on the findings of this project--this protocol, if successfully developed, will allow iterative transformation to insert multiple genes into the same locus.
Impacts
What was accomplished under these goals?
In the third year of the project, we studied FLP-FRT mediated site-specific integration (SSI) and Cre-lox mediated marker excision in two additional target lines. In the previous years, we carried out a pilot study using two FRT (target) lines, A and B. To evaluate whether previous findings are more general or specific to target lines A and B, we included two more target lines, C and D, in this year of the project. Using gene gun, we first developed SSI lines in each target lines. SSI development invloves gene gun mediated DNA delivery (co-bombardment of donor vector with FLPe gene) into embryo-derived callus and selection on geneticin to isolate SSI lines [Nandy and Srivastava (2011) Plant Biotech J. 9:713-721]. We generated 12 SSI lines with target line C and 6 SSI lines with target line D from 40 explants, giving and efficiency of 30 -15%, respectively. A similar efficiency (10 - 30%) was obtained with target lines A and B. Then we selected plants from 4 SSI lines (2 each from C and D parents: C.1, C.2, D.1, D.2), heat-shocked them using the protocol given earlier [Nandy and Srivastava (2012) Plant Biotech. J. 10:904-912] for marker excision. These plants were allowed to grow for 2 weeks then a leaf section was taken for PCR analysis. A total of 6 SSI plants were subjected to heat treatment (1 plant each of C.1 and C.2, and 2 plants each of D.1 and D.2). PCR revealed that heat-treatment was not effective in excising marker genes from C.1; however, plants of the remaining lines showed complete excision of the marker genes. Each analysis involved non-treated control plants where marker gene was duly maintained. Heat-treated plants and the non-treated controls have been moved to greenhouse to grow and set seeds. Progeny analysis is awaited.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2011
Citation:
Srivastava V, Akbudak MA, and Nandy S (2011) Marker-Free plant transformation. In: Plant Transformation Technology: Revolution in Last 27 years. (Eds: Y. Dan and D. W. Ow). Chapter 6.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Srivastava V (2013) Site-Specific Gene Integration in Rice. Rice Protocols, Methods in Molecular Biology 956: 83-93.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Akbudak MA, Nicholson SJ, and Srivastava V (2013) Suppression of Arabidopsis genes by terminator-less transgene constructs. Plant Biotechnol. Rep. DOI 10.1007/s11816-013-0278-z
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Srivastava V. A robust marker-free site-specific gene integration technology. Abstract. Proceedings of Plant & Animal Genome meeting, San Diego, CA, Jan 12-16, 2013
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
Srivastava V. Application of FLP-FRT System for Site-specific Gene Integration in Rice. Abstract. Proceedings of Society of In Vitro Biology, Seattle, WA, June 15-19, 2012.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: Using two target lines (A and B), multiple site-specific integration (SSI) lines were developed (>10). FLP-FRT recombination was utilized for SSI of a GUS gene. Each of these SSI lines was analyzed by PCR and Southern hybridization to isolate precise integration of a single copy of the transgene (GUS). The SSI locus was equipped with heat-inducible Cre activity and strategically located loxP sites to remove all DNA (including marker genes) flanking GUS gene. SSI lines (young plants) were heat-treated at 42 C for 3 hours to induce cre activity, and then allowed to grow normally. DNA from adult plant was isolated to analyze marker excision. This analysis indicated that Cre-mediated DNA excision was extremeley efficient with none or only trace amount of marker genes detectable. T1 seeds borne on these lines were germinated and analyzed on Southern blot for locus structure. This analysis revealed that all lines transmitted marker-free SSI (MFSSI) locus (fully excised) to the next generation at a very high efficiency. This data indicated that FLP-FRT mediated SSI and heat-induced Cre mediated marker excision is a robust approach for generating marker-free SSI locus, in which gene-of-interest flanked with two oppositely oriented loxP sites is present. Finally, we analyzed transgene expression before and after marker-excision, and found that transgene activity is not altered through the marker-excision process. Thus stable transgenic lines are produced by this novel transformation platform. PARTICIPANTS: 1) Soumen Nandy, Postdoc 2) M. Aydin Akbudak, postdoc 3) Vibha Srivastava, P.I. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project developed and validated the molecular approach for directing precise transgene integration into a specific genomic site, and then removing marker genes to generate a 'clean' transgenic line. The developed method/ approach is robust, and the transgenic lines produced in this method display transgene stability.
Publications
- Nandy S and Srivastava V (2012) Marker-free site-specific gene integration in rice based on the use of two recombination systems. Plant Biotech. J.10: 904 - 912.
- Nandy S and Srivastava V (2011) Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system. Plant Biotech J. 9: 713-21.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: The overall goal of this project is to develop precise plant transformation method consisting of marker gene removal from site-specific integration locus. The project utilizes rice as the model for plant transformation. A molecular strategy for this method was proposed, which involves following experimental steps: (1) Develop transgenic lines using FLP/FRT recombination to drive site-specific integration, (2) Heat-induce cre gene (incorporated in the locus)to initiate excision of selection marker genes. (3) Test the utility of dhlA gene as a negative selection marker for improving the efficiency of the clean locus recovery. In past 5 months, site-specific integration (SSI) lines of rice have been developed that will be used for testing marker excision in subsequent period of this project. The SSI lines are derived from from two founder lines A and B (also called as FRT-target lines). A total of 3 SSI lines from A line and 8 from B line are available. using Southern analysis, single-copy (SC) SSI lines have been identified. So far 4 SC B-SSI lines have been identified. While multi-copy SSI lines will also be used for marker-excision experiment, analysis of SC lines will be much easier. So far, T0 plants of these lines have been generated that are growing in greenhouse. T1 seed set is awaited. Heat-treatment (42 C for 3-6 hours) on a sample of these lines has also been done. PCR analysis using genomic DNA from the treated tissue revealed that excision of lox-flanked marker genes is occurring as a result of cre-induction. PARTICIPANTS: 1. Soumen Nandy, postdoc 2. M. Aydin Akbudak, postdoc TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The data available in past 5 months support the functionality of the molecular tools applied in the proposed technology. However, these data are too preliminary to assess the impact of the research. The project involved one postdoc.
Publications
- 1. Akbudak MA and Srivastava V (2011) Improved FLP recombinase, FLPe, efficiently removes marker gene from transgene locus developed by Cre-lox mediated site-specific gene integration in rice. Mol. Biotechnol. 10.1007/s12033-011-9381-y.
- 2. Khattri A, Nandy S, and *Srivastava V (2011) Heat-inducible Cre-lox system for marker excision in transgenic rice. J. Biosciences 36(1): 1-8.
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