Progress 10/01/07 to 09/30/13
Outputs
Target Audience: Not relevant Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project trained one two postdoctoral fellows, Drs. Soumen Nandy and M. Aydin Akbudak, and one graduate student, Mr. Linh Nguyen. These participants of the project attended annual conference of the Society of In Vitro Biology at Seattle, WA in June 2012, and presented their work. Drs. Nandy and Akbudak presenated a talk, and Dr. Akbudak won 3rd place in postdoc competition. Mr. Nguyen presented a poster. Dr. Akbudak has taken a position as Assistant Professor in Turkey, and Mr. Nguyen has returned to Vietnam to a position of Plant Tissue Culture lab in-charge. Dr. Nandy is continuing with my group as a postdoctoral associate in the ongoing rice biotech work How have the results been disseminated to communities of interest? Results of this project have been presented in the scientific conferences such as annual SIVB and PAG meetings. Additonally, journal publications, and book chapters have been published. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall goal of this project is to develop a new method of plant transformation in which precise, marker free transgene locus can be made repeatedly. We employed FLP-FRT system for site-specific gene integration in rice (Nipponbare background), and Cre-lox for marker excision from the locus. In the initially phase of this project, we developed 4 target (FRT) lines called, A, B, C and D. We developed site-specific integrations (SSI) mostly in A and B lines, and studied the efficiency of gene gun mediated SSI and heat-shock induced marker-excision. The latter step (marker-excision) was induced by heat shock treatment as the heat-inducible Cre gene was utilized for streamlining the process. We found that SSI efficiency was 10 - 30% in A and B, respectively. However, marker-excision ocurred at at high rate: mostly at >90%. Excision efficiency was defined as number of T1 progeny containing marker-free locus (not counting the non-transgenic progeny due to transgene segregation). In the last year, we focused on two additional target lines C and D. Again we obtained SSI at a similar rate (30% in target line C , and 15% in target line D). Then we selected plants of all 4 SSI lines (2 from each of C and D target line) for the efficiency of marker excision. These 4 SSI lines are named, C.1, C.2, D.1 and D.2. For this purpose, young regenerated shoots (in glass tubes) were incubated at 42 degree celcius for 3 hours (heat-shock), and this treatment was repeated 3 times for 3 consecutive days. Between treatments, plants were kept at at room temeprature. These plants were allowed to grow for 2 weeks in the tubes. After 2 weeks, a small leaf section was taken for DNA isolation and PCR to analyze marker excision. A non-heat-shock control (clones of the same line) were also included in the analysis. A total of 6 plants were subjected to heat-shock treament (1 plant each of C.1 and C.2, and 2 each of D.1 and D.2). PCR revealed that heat-shock treatment was not effective on C.1 plant, but it removed marker gene from all remaining plant lines. These plants were moved to greenhouse for seed set. T1 analysis is awaited.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Akbudak MA, Nicholson SJ, Srivastava V (2013) Suppression of Arabidopsis genes by terminator-less transgene constructs.
Plant Biotechnol. Rep. 7: 415-424
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Srivastava V. A robust marker-free site-specific gene integration technology. In Annual Plant & Animal Genome Conference, San Diego, CA, Jan 12 -16, 2013.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Srivastava V. Application of FLP-FRT System for Site-specific Gene Integration in Rice. In Annual conference of the Society of In Vitro Biology, Seattle, WA, June 3 - 7, 2013.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2012
Citation:
Linh Nguyen (2012) Evaluation of the recombination efficiencies of FLP proteins. M. S. thesis submitted to University of Arkansas
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The goal of this project is to develop "clean" and "precise" plant transformation method. The precision in gene integration is imparted by the use FLP-FRT recombination system that directs the integration of foreign DNA into the predetermined genomic location (founder lines) without making "unexpected" changes in the genomic site. The locus is then cleaned out of selection marker gene by inducing Cre-lox recombination, which precisely deletes the marker gene. In previous years, this strategy was developed and demonstrated on two founder rice lines. In 2012, the strategy was tested on additional two founder lines that carry "target site" for FLP-FRT mediated transgene integration. The transformed callus lines containing site-specific integration of transgenes were obtained from each founder line at roughly the same efficiency (1 transformant per bombardment). The transgenic lines were then subjected to heat-treatment to induce Cre-lox system, which resulted in the deletion of the marker gene at rates ranging from 20 - 100% (% of cells without marker gene as determined by PCR). Together these experiments validated the "marker-free site-specific gene integration" approach on a total of four founder lines. Additionally, experiments were done to analyze the effect of marker-deletion on the gene-of-interest (GUS gene). Progeny plants containing marker-free locus were compared with their marker-containing siblings for GUS expression. This experiments revealed that there was no apparent effect of marker-deletion on the expression of GUS gene. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The proposed method of "clean" and precise genetic transformation has been tested on four different rice lines. In each case, transgenic lines were obtained at an efficiency similar to that of the conventional approach (30 - 50%). However, the advantage here is that the transgenic locus could be rapidly converted to marker-free locus. In addition, the transgene locus developed by this approach contains minimal or no "unexpected" mutations, which are commonly found in the transgene locus developed by conventional transformation approach. These unexpected mutations could be deletions, additions or even duplications at the transgene locus. Thus, this project developed a more precise method of plant transformation. The method was experimentally demonstrated on rice; however, it is applicable on other plant species.
Publications
- Srivastava V (2013) Site-Specific Gene Integration in Rice. In: Rice Protocols, Methods in Molecular Biology 956: 83-93. (Ed: Y. Yang).
- Nandy S and Srivastava V (2012) Marker-free site-specific gene integration in rice based on the use of two recombination systems. Plant Biotechnol J. 10(8):904-12.
- Srivastava V, Akbudak MA, Nandy, S (2011) Marker-Free Plant Transformation. In: Plant Transformation Technology: Revolution in Last 27 years. (Eds: Y.Dan and D. W. Ow).
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: In 2011, we developed a method for "clean" transformation using rice as the model crop. In this method, precise transgene integration was followed by the removal of marker genes to develop "clean" transgene locus. Specifically, we used FLP-FRT system for integrating a gene cassette, and then induced marker-excision mediated by Cre-lox system. The marker-free precise transgenic plants were obtained in the first generation that transmitted marker-free transgene locus into next generation at 100% efficiency. Ten site-specific integration lines of rice were developed, 4 of which were precise single-copy lines. Within the locus a heat-inducible Cre gene was incorporated to induce marker excisions. One-two week old regenerated plants of the four lines (in glass tubes) were incubated in 42 C water bath for 3 hours to induce Cre activity. The procedure was repeated 2-3 time every 24 hours. The plants was then transferred to soil and allowed to grow and set seeds. Southern analysis on three lines showed complete excision of marker gene, while the fourth one was incomplete. Accordingly, 100% of progeny derived from the "completely excised" plants inherited 'marker-free' locus, while only 22% of the progeny derived from "incompletely excised" plants showed marker excision. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Presence of undesirable genes in transgene locus is a major concern, in addition, transformation process based on random integration mechanism may incorporate mutations into the genome. To eliminate these undesirable features associated with conventional transformation process, the present project used recombinase mediated gene integration approach followed by recombinase mediated marker excision. The resulting transgene locus is precise and free of marker genes.
Publications
- No publications reported this period
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: In 2010, we developed a method of using FLP-FRT recombination system for precisely integration foreign genes in rice genome. Previously, this technique was shown using Cre-lox recombination system, but for more flexibility, it is important to be able to perform gene integration using alternative recombination systems such as FLP-FRT. We demonstrated that a DNA construct containing FRT sites (Donor construct) can recombine in rice cells in the presence of FLP protein and integrate into previously inserted FRT site. The limiting step in this process was identified as the FLP recombinase activity. When FLP gene is expressed from the target locus that contains a FRT between promoter and gene coding region, FLP activity is very low. That necessitates the co-introduction of FLP gene with the donor construct. We used a thermostable variant of FLP protein, called FLPe, to drive site-specific integration. These experiments generated multiple site-specific integration lines that carried the precise integration locus as predicted from the FRT x FRT recombination. The integration locus was faithfully transmitted to next generation. Thus, FLP-FRT mediated site-specific gene integration was demonstrated in rice genome. PARTICIPANTS: 1. Soumen Nandy, Postdoc 2. M. Aydin Akbudak, Graduate student 3. Jamie Underwood, Program Technician TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The FLP-FRT mediated site-specific gene integration method is needed for developing marker-free site-specific gene integration approach. In this approach, site-specific gene integration is obtained by using FLP-FRT system and selection marker gene is subsequently removed by using Cre-lox recombination.
Publications
- 2. Nandy S and *Srivastava V (2010) Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system. Plant Biotech J. doi: 10.1111/j.1467-7652.2010.00577.x
- 3. Khattri A, Nandy S, and *Srivastava V (2011) Heat-inducible Cre-lox system for marker excision in transgenic rice. J. Biosciences (in press).
- 4. Srivastava V, Akbudak, MA, Nandy, S (2011) Marker-free plant transformation. In: Plant Transformation Technology Revolution in Last Three Decades. Dan and Ow (eds). Bentham Science Publishers (in press).
- 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.
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The goal of the project is to develop precise transgene integration system. We used site-specific recombination systems, Cre-lox and FLP-FRT for this purpose. We found that multiple copies (1-3) of a transgene can be integrated precisely into rice genome using Cre-lox system. Each transgene expressed properly as indicated by the dosage dependent expression pattern. Next, we found that FLP-FRT system is quite inefficient in for gene integration, but certain modifications can be made to bring up its efficiency to an acceptable level. We found that presence of a FRT site between promoter and FLP gene down-regulates FLP activity severely. On the other hand, Cre-lox works efficiently in a similar design. Thus we modified the integration strategy that included background expression (transient or stable) of FLPe gene, an enhanced version of FLP gene. With this strategy, we obtained several site-specific integration events. PARTICIPANTS: 1. Soumen Nandy, postdoc 2. M. Aydin Akbudak, Graduate student 3. Jamie Underwood, Program Associate TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1. This study shows that site-specific gene integration approach is suitable for engineering multigene locus. 2. This study developed a strategy for FLP-FRT mediated site-specific gene integration.
Publications
- 1. 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
- 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. DOI: 10.1007/s12033-009-9235-z
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: With the goal of developing 'clean' and precise transformation technologies, recombinase mediated site-specific integration and marker gene excision are being tested. The molecular strategy of this technology involves the use of two separate recombination system. We chose Cre-lox, which has been validated to work efficiently, and FLP-FRT. The FLP-FRT system is the second most utilized recombination system in plants but its effieincy is not well established. In the last year, we developed heat-shock Cre and heat-shock FLP lines of rice and found that the FLP lines are mostly inefficient in carrying out excisional recombination. We also tested the integration recombination using FLP-FRT system, and again found it to ineffective in catalyzing an integration reaction. Therefore, most of the efforts were directed in finding ways to improve the efficiency of FLP-FRT system for transgene integration in rice. PARTICIPANTS: 1. Soumen Nandy, Postdoc, developed rice transgenic lines, and carried out integrative bioassays to compare Cre-lox with FLP-FRT. 2. M. Aydin Akbudak, Graduate Student, developed clean integration lines, and compared FLP with FLPe and FLPo. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Based on the findings that FLP-FRT system is mostly ineffective in rice in carrying out integrative integrations, alternative approaches have been developed. First, we are testing is the co-expression of improved FLP genes, FLPe or FLPo will boost FLP activity far enough to drve integration. Additional recombination systems will be tested that include phiC31 and R-RS.
Impacts
1. Expression of FLP is highly compromised when an FRT site is present between its promoter and the coding region. 2. Expression of a Cre gene from a construct containing a lox site between promoter and coding region is sufficient to carryout both excisional and integrative recombinations. 3. While FLP recombiase carries out a complete reaction, its specific activity is far lower compared to that of Cre in excisional recombination. 3. In a bioassay based on testing the integrative recombination between two FRT or lox sites catalized by their respective recombinases (FLP or Cre), high number of lox-integrations were ontained, while no FRT-integrations were obtained. 4. In a transient assay based on excisional recombination on extrachromosomal plasmids, improved FLP genes called FLPe and FLPo were found to be 2-3 times more efficient than the original FLP gene.
Publications
- 1. Srivastava V, Gidoni D, and Carmi N (2009) Site-specific gene integration technologies for crop improvement. In vitro cellular and developmental biology - Plant (in press).
- 2. Gidoni D, Srivastava V and Carmi N (2008) Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants. In vitro cellular and developmental biology - Plant 44: 457-467.
|
Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The goal of the project is to develop a streamlined method for highly stable expression of foreign gene (transgene) in rice from a 'clean' integration locus. 'Clean' locus will contain the gene-of-interest (GOI) only; and therefore, marker genes will have to be removed. This project is an extension of our previous project, wherein, we found that a highly stable and predictable level of gene expression is obtained from a precise transgene locus engineered by Cre/lox-mediated site-specific recombination. Therefore, we propose to add marker-excision tool into the precise transgene integration technology to generate a clean-site-specific integration technology. Secondly, we ptopose to determine if expression from a site-specific integration locus can be boosted up by increasing the gene copy number. Finally, we want to determine whether a bacterial dehalogenase A gene (dhlA) can be used as a negative selection marker in rice callus cultures. The rationale of these objectives are
(1) clean integration will enhance acceptability of transgenic products, (2) boosting gene expression will be useful for production of pharma proteins in plants, (3) a negative selection marker will be useful for enriching rice callus for marker-free cells.
PARTICIPANTS: Vibha Srivastava: Principal Investigator Sarah K Moore: Research Technician Anjali B. More: Research Technician Soumen Nandy: Postdoc Aydin Akbudak: Graduate student
Impacts
First objective: to develop clean-site-specific integration: Different components of a molecular strategy, developed earlier, were tested. We used two separate approaches: first approach consisted of Cre/lox-mediated gene integration followed by heat-inducible-FLP-mediated excision. As we already had plasmid constructs, we made much more progress in this experiment than in the experiment based on the reverse approach. Here, we found that Cre/lox recombination system can be used in conjunction with heat-inducible FLP/FRT system to develop integration locus containing FRT-flanked marker genes. Thus marker genes are primed for excision by FLP activity. For the reverse approach consisting of FLP/FRT-mediated integration followed by heat-inducible-Cre-mediated excision of lox-flanked marker genes, we developed vectors, which are being used for transformation of rice. One vector (target) contains between two inversely oriented lox sites, FLP/FRT system to carryout
site-specific gene integration and heat-inducible cre gene. The second vector (integration) contains a lox-flanked GOI and FRT sites to facilitate the proper integration of the gene into a target FRT site. These vectors are ready for transformation of rice. Second Objective: Effect of gene copy number on expression level: We found that by increasing gene copy number from 1 to 2 or 3, expression increased 2-4 fold. Additionally, we found that use of 35S promoter is unfavorable to gene expression. Specifically, we found that when maize ubiquitin promoter driven GUS gene is integrated, no silencing is observed among 125 site-specific integration lines. Whereas, 35S-driven GUS gene becomes silenced in 25% of the site-specific integration lines (3 out of 12). Third objective: Negative selection marker for rice callus: We tested the ability of dhlA gene to serve as negative selection marker in rice callus. We found that fumigation of rice callus expressing dhlA gene with 100 microliters of
dichloroethane (DCE) is sufficient to arrest callus growth for 2 weeks.
Publications
- 1. Moore SK, Srivastava V. 2008. A bacterial haloalkane dehalogenase (dhlA) gene as conditional negative selection marker for rice callus cells. In Vitro-Plant (provisionally accepted).
- 2. Gidoni D, Srivastava V, and Carmi N. 2008. Site-specific excisional recombination approaches for agricultural biotechnology. In Vitro Cellular and Developmental Biology-Plant (in press).
- 3. Srivastava V (2007) Site-specific recombination for precise and clean transgene integration in plant genome. Plant Transformation Technology Conference, Vienna, Austria, Feb 3 - 7, 2007.
|
Progress 01/01/06 to 12/31/06
Outputs
In the previous years while developing site-specific integration technology, we found that not only gene expression (from a precise locus) is stable and predictable, it doubles when a plant becomes homozygous. Further, the doubled gene expression level is inherited through successive generations. Several studies on stability of transgene expression have suggested that gene expression over a certain threshold is subject to gene suppression or silencing. So we decided to test whether gene expression from allelic position can increase with gene dosage. Towards this we developed transgene vectors containing one copy, two copies or three copies of 35S-GUS or 35S-GFP gene. Each of these vectors were bombarded into the target cell lines, where they are expected to integrate site-specifically in a known genomic site. The cell lines containing 1-3 dosage of each gene will be tested for gene expression level to address the expression-threshold of the locus. The demonstration of
increase in expression level with increasing gene dosage is required to develop a technology for producing pharma-proteins in a transgenic platform. Towards this concept, we developed 35S-GUS and 35S-GFP constructs representing 1-3 gene copies. Site-specific integration of these constructs into a well characterized rice genomic site is underway.
Impacts
Develoment of site-specific integration technology will be useful for the overproduction of certain proteins in plants. This technology will be especially useful for production of pharmaceutical proteins in plant cell cultures.
Publications
- Vibha Srivastava and Scott J. Nicholson (2006) Cre/lox technologies for plant transformation. CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources 1, No. 034
|
Progress 01/01/05 to 12/31/05
Outputs
The goal of the project is to develop a streamlined method for transgenic plant production. The strategy is based on engineering a precise (site-specific) transgene locus by utilizing Cre/lox recombination system. In previous years we reported the efficiency and efficacy of Cre/lox-mediated precise genetic engineering in rice as a model system. This technology was found to be efficient for generating stable site-specific integration transgenic rice at high efficiency. In 2005, we carried out progeny analysis on 18 different transgenic lines containing site-specific integration of GUS gene. We found that GUS locus in each line was inherited consistently through successive generations; and homozygous plants contained twice as much GUS activity level as hemizygous plants. This allelic gene dosage is a hallmark of transgene stability. Next, we attempted on developing a breeding method for obtaining site-specific gene integration. Since the technology relies on using a
specific DNA delivery method (particle bombardment-mediated DNA transfer), we attempted on developing this alternative approach. Agrobacterium mediated gene transfer is a common transformation method; however, it is considered inefficient for Cre/lox-mediated gene integration. Therefore, we studied if we can obtain Cre/lox-mediated site-specific gene integration by crossing in two unique transgene locus (which can be developed by any transformation method), target and donor loci. Target locus contained the insertion site and cre gene, while donor locus contained multiple copies of the 'transgene-of-interest' (TOI). Cre-mediated recombination in the donor locus was expected to loop out multiple copies of TOI and direct their insertion into the target locus. To test this hypothesis we crossed a target line with two independent donor lines, and analyzed their F1 and F2 progenies. We found that in F1 plants of both crosses, Cre-mediated recombination was initiated at various developmental
stages resulting in looping out of TOI. Next, we analyzed F2 plants to determine if any of the looped-out copies re-integrated in the genome. This analysis revealed that in both crosses all TOI copies were completely lost i.e. they neither integrated into target locus nor elsewhere. The data suggests that site-specific integration technology will continue to rely on particle bombardment mediated gene transfer
Impacts
Development of streamlined method for producing transgenic rice
Publications
- Moore SK and Srivastava V (2006) Efficient deletion of transgenic DNA from rice mediated by Cre/lox recombination system. Crop Sci 46:700-705
- Chawla R, Ariza-Nieto M, Wilson AJ, Moore SK and Srivastava V (2006) Transgene expression produced by biolistic mediated site-specific integration is inherited consistently through subsequent generations. Plant Biotech J 4:209-218
|
Progress 01/01/04 to 12/30/04
Outputs
We generated over 50 site-specific integration lines in 2004 that contained precise integration of GUS gene into a pre-selected genomic location. These transgenic lines were subjected to molecular analysis (PCR and Southern blotting) to detemine the presence or absence of precise integration locus and illegitimate integrations in the background. If only precise integration occurred the transgenic lines were classified as single-copy (SC) and if illegitimate integrations were found (in addition to the precise locus) in the genome, the line was classified as multi-copy (MC) line. Selected SC and MC lines (18 in total) were subjected to gene expression analysis over successive generations. The data validated the observations that were made in the previous year. These are: 1. Gene expression variability can be significantly reduced by placing the foreign gene precisely into a pre-dertermined genomic location. 2. The transgenic lines containing precise integration locus
expresses GUS gene consistently over successive (3 tested) generations. 3. Illegitimate interations often cause gene silencing of GUS gene in the precise integration locus. These integrations can either be genetically linked to or segregated from precise integration locus. 4. Upon segregation of illegitimate integrations, silenced GUS gene is activated to the levels consistent to the genomic position.
Impacts
This project may lead to the development and evaluation of a technology for streamlining the production of transgenic plants.
Publications
- Radhakrishnan P and Srivastava V (2004) Utility of the FLP-FRT recombination system for genetic manipulation of rice. Plant Cell Rep. (in press, available online DOI: 10.1007/s00299-004-0876-x).
- Srivastava V and Ow DW (2004) Marker-free site-specific gene integration in plants. Trends in Biotechnology 12:627-630.
- Srivastava V, Ariza-Nieto M and Wilson A (2004) Cre-mediated site-specific gene integration for consistent gene expression. Plant Biotech. J. 2:169-179
|
Progress 01/01/03 to 12/31/03
Outputs
This project is aimed to develop a method for streamlining the production of transgenic plants. Transgenic plants produced by traditional techniques have to be screened extensively to find a stable breeding line. The high level of variation observed between tranegnic plants has been attributed to the process of random integration in undertermined pattern. Therefore, site-specific gene integration holds promise for reducing the variation among transgenic lines and generating consistent levels of gene expression. Site-specific gene integration can be obtained by several different DNA delivery methods. In the present project, we proposed to evaluate the efficiency and effectiveness of biolistic mediated site-specific gene integration in producing transgenic rice with consistent expression levels. Towards this we developed a molecular strategy that involves the use of a site-specific recombination system, Cre-lox. First, a single lox site is placed in a random genomic
location, which serves as the insertion (target) site for site-specific integration step. We developed 32 precise integrant lines using a target line called, T5; 14 using target line, T43; and 3 using T68 ( all in Taipei 309 background). We also developed 3 new target lines called N19, N20, N22 in Nipponbare background. All site-specific integrant lines were subjected to Southern blot analysis and PCR to determine whether or not the transgene locus was precise. First, we removed imprecise lines from the population, then we classified precise integrant lines into two categories: single copy (SC) and multi-copy (MC). Multi-copy lines were recovered because in addition to site-specific integration, random integrations also occurred in some cases. Later, we studied the expression level of reporter gus gene in both SC and MC lines. First, quantitative GUS assay was done using callus of the integrant lines. We found that SC lines show more or less consistent level of gene expression. The
variation of GUS activity between different SC lines was only 2.5 fold. However, MC lines showed higher variation (0-100) and lower expression than SC lines. Then we attempted to regenerate plants from these callus lines, only 5 SC and 3 MC lines (T5 derived) were regenerable. GUS assay using leaf tissue of these lines showed that leaf cells express gus gene at more or less same level as callus cells. Further, GUS was done on T1 seedlings and we found that GUS activity is consistent in T1 plants. This data demonstrates that biolistic mediated site-specific integration method is useful for rapid production of stable transgenic lines and therefore suitable for streamlining the production of transgenic plants. We have developed a follow up strategy that can preclude random integrations. This strategy relies on the use of negative selection marker to deselect multi-copy lines. We chose codA (cytosine deaminase) for this purpose. Our experiments indicate that codA gene is either
ineffective in rice or we have undetected mutation in our DNA construct. We have not found an alternative negative selection marker and therefore shelved this objective.
Impacts
This project will lead to the development and evaluation of a technology for streamlining the production of transgenic plants.
Publications
- Srivastava V, Ariza-Nieto M, Wilson AJ (2004) Cre-mediated site-specific gene integration for consistent transgene expression in rice. Plant Biotech. J. (in press).
|
Progress 01/01/02 to 12/31/02
Outputs
We are developing a technology for targeted gene integration in rice, which will be useful for generating stable transgenic plants. Following is the progress: 1. Efficiency of site-specific gene integration: (a) Generation of site-specific transgenic lines: We are using a Cre-lox based strategy for obtaining precisely targetd transgenic plants. To study the efficiency of this strategy, we have used an existing target line, t5. First, we generated t5 callus tissue, and then we delivered the donor DNA that contains a gene of interest (gus) using biolistics. We have carried out 16 different experiments of 10 bombardments each. At present, 5 of these experiments have been completed and the rest are underway. We have observed that the efficiency varied in each of the 5 experiments from 10% to 100%, where efficiency is reported as number of transgenic lines per bombarded plate. With a total of 24 targeted lines out of 50 bombardments, the average efficiency comes out to be
50%. (b) Molecular Analysis: These 24 integrant lines represent a pool of single copy lines (stable lines, suitable for breeding programs), and multi-copy lines (unsuitable for breeding). So the next step was to determine the number of single copy integrant lines in this pool, which was done by Southern analysis. We found that 13 out of 24 (50%) were single copy. (c) Summary: Using biolistic method of DNA delivery, the strategy works at 25% efficiency (transgenic lines/ bombardment) for obtaining precise integrant lines. 2. Efficacy of the Molecular Strategy (a) Transgene expression in the integrant lines: An expectation of the integrant (single copy) lines is that they will express gus gene at higher and similar levels, whereas multi-copy lines will display lower level of expression and higher variation between lines. To study this we have done quantitative GUS assay with 8 single copy and 8 multi-copy integrant lines. The data demonstrates that majority of the multi-copy lines
contain 4x lower GUS activity as compared to single copy lines. Also the variation between lines was 3x higher in multi-copy lines as compared to the same in single copy line. (b) Summary: The GUS data on integrant lines shows that higher percent of single copy lines express gus gene at similar level, and that the majority of multi-copy lines display lower gene expression and higher variation. This data suggests that the molecular strategy is effective for generating stable transgenic lines.
Impacts
The data has demonstarted that the strategy under test is effective in obtaining the goals of the project i.e. developing a method for generating stable transgenic rice plants. Transgenic plants produced using this strategy will be stable over generations and therefore suitable for breeding programs. Less time and money will need to be expended for characterization of the transgenic plants produced by this method.
Publications
- No publications reported this period
|
|