The authors that wrote this review article are David Gidoni1 (left), Vibha Srivastava2 (center), Nir Carmi1 (right).
1Institute of Plant Sciences, Agricultural Research Organization Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel
2Department of Crop, Soil & Environmental Sciences, University of Arkansas, Fayetteville, AR, USA
Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants
A major limitation of crop biotechnology and breeding is the lack of efficient molecular technologies for precise engineering of target genomic loci. The random introduction of complex transgenic DNA into the plant genome, as implicated by current direct- and Agrobacterium-mediated transformation methods, generates unpredictable effects on both transgene and homologous native gene expression. The environmental risk of transgene transfer into related plant species and consumers is another concern associated with these conventional transformation technologies. Various approaches to avoid or eliminate undesirable transgenes, most notably selectable marker genes used in plant transformation, have recently been developed. These approaches include co-transformation with two independent T-DNAs or plasmid DNAs followed by their subsequent segregation, transposon-mediated DNA elimination, and most recently, attempts to replace bacterial T-DNA borders and selectable marker genes with sequences of plant origin that exhibit equivalent functions. Comparatively, the use of site-specific recombination to remove undesired DNA from the plant genome and concomitantly, via excision-mediated DNA rearrangement, switch-activate by choice transgenes of agronomical, food or feed quality traits provides a powerful more versatile “transgene maintenance and control” strategy. This strategy can significantly contribute to the transfer of transgenic laboratory developments into farming practice. These three laboratories’ (David Gidoni, Vibha Srivastava, Nir Carmi) main goal is development of site-specific excision/integration recombination strategies, to improve the control and management of transgene DNA in plants, in a stable, environmental and consumer friendly manner. Here, in this collaborative review article we focused on recent reports demonstrating the elimination of undesirable transgenes (essentially selectable marker and recombinase genes) from the plant genome and concomitant activation of a silent transgene (e.g., a reporter gene) mediated by different site-specific recombinases driven by constitutive or chemically, environmentally or developmentally regulated promoters. These reports indicate major progress in DNA/gene excision strategies which extends application of the technology from annual, sexually propagated plants towards perennial, woody and vegetatively propagated plants. Current trends and future prospects for optimization of excision-activation machinery and its practical implementation for the generation of transgenic plants and plant products free of undesired genes are discussed.
David Gidoni, Vibha Srivastava, and Nir Carmi. Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants, In Vitro Cellular & Developmental Biology-Plant 44:457–467, 2008.
|Pictured left to right are first row: Brianna Prante, Kiera Garman, second row: Dr. Brandon Sims and Dr. Suzanne Lindsey
Matrix-coated transwell cultured TM4 Sertoli cell testosterone-regulated gene expression mimics in vivo expression
Research performed in our laboratory addresses cell-specific gene expressions that potentially play roles in activities and functions of that cell type. Spermatogenesis is a complex process that is not fully understood. During this developmental process in the adult male testes, many phases with complicated mechanisms at each step take place. Sertoli cells that reside in the seminiferous tubules of the testes, possess androgen receptors, which bind testosterone and activate specific testosterone regulated transcripts. Testosterone signaling, required for spermatogenesis to take place, modulates reassembling and disassembling of cell-cell contacts, which is essential for development of viable spermatozoa. Without testosterone, these cell-cell contacts permanently breakdown and the loss of developing spermatids occurs. Detailed mechanisms of how this occurs in vivo has been hampered by a lack of in vitro methods by which in vivo-like testosterone signaling can be studied. As an example, Sertoli cell-specific Pem, also known as Rhox5, homeobox gene expression has not been previously induced in vitro by testosterone treatment even though this occurs in vivo. In our recently published research of the TM4 mouse Sertoli cell line, we show that culturing on a basement membrane-like material in a bichamber system in which testosterone can be added to the bottom chamber induced similar gene expressions, including Pem/Rhox5, to those found in testosterone regulated phases of cross-sectioned seminiferous tubules. Further use of this and other similar cultures of androgen receptor positive cell lines can potentially elucidate causes of male infertility that are yet unknown.
Brianna C. Prante, Kiera L. Garman, Brandon N. Sims, and J. Suzanne Lindsey. Matrix-coated transwell cultured TM4 Sertoli cell testosterone-regulated gene expression mimics in vivo expression, In Vitro Cellular & Developmental Biology-Animal 44:434-443, 2008.
Induction of hairy root cultures from Gossypium hirsutum and Gossypium barbadense to produce gossypol and related compounds
This project is a collaborative effort between two research units at the USDA-ARS Southern Regional Research Center and the Department of Biology at the University of New Orleans and began as an undergraduate independent-research project by Ms. Stephanie Moss. For many years, scientists in the Commodity Utilization Research Unit have collaborated with international groups seeking novel uses for gossypol, a yellow pigment found in cotton seeds, roots, leaves, and stems. Finding new applications for this sesquiterpenoid compound will enhance the value of cotton seeds, a by-product of cotton fiber processing. Thus far, gossypol has been found to suppress numerous microbes, parasites, fungi, and insects. Also, there are several clinical trials using gossypol to inhibit the growth of some cancers. In order to have a year-round supply of plant material for gossypol extraction and to determine if the ratios of gossypol chiral isomers could be altered in vitro, our team initiated a project to determine if cotton hairy roots would produce gossypol.
Transformation with Agrobacterium tumefaciens is notoriously dependent on the cotton genotype used, but transformation of both accessions of two cotton species with Rhizobium rhizogenes proved successful immediately. With the particular strain of R. rhizogenes used for these experiments, young hypocotyls sections were more effective than leaf sections in producing viable hairy roots. Although a small amount of gossypol was found in culture media, the majority of gossypol produced was associated with the hairy root tissue. Remarkably, the levels of gossypol produced by hairy roots often exceeded the levels normally found in cotton seed. Extraction of gossypol from cotton seed is often complicated by the presence of large amounts of oil; however, this complication does not exist for the hairy root cultures. Most importantly, individual transformants produced gossypol at fairly stable levels for more than two years. Although the research project was interrupted by Hurricane Katrina, our team has optimized culture conditions for inducing hairy roots from two cotton species that produce high levels of gossypol and related compounds. Our cultures have also been used by numerous investigators studying nematode-plant interactions. The team is currently investigating the effects of elicitors to boost gossypol production to even higher levels.
Barbara Triplett, Stephanie Moss, John Bland, and Michael Dowd. Induction of hairy root cultures from Gossypium hirsutum and Gossypium barbadense to produce gossypol and related compounds, In Vitro Cellular & Developmental Biology-Plant 43:508-517 2008.
An Innovative Method for Cell Line Identification and Interspecies Cross-contaminations: Cytochrome b Polymerase Chain Reaction-Restriction Fragment Length Polymorphism analysis
The Cell Culture Centre in Brescia is one of the largest cell culture collections in Italy and, currently, collects about 600 cell culture types, corresponding to over 40,000 frozen ampoules. These cells can be subdivided into established “normal” cell lines, established tumoral cell lines, hybridomas and primary cell cultures (including adult stem cells from different animals and tissues) belonging to more than 30 animal species. Together with the preparation, amplification and storage of all cell cultures a key role is played by the quality controls. In fact, in our laboratory, these controls are systematically performed during each step of the manipulation process with the aim to verify not only the microbiological and virogical contamination and the tumorigenicity properties, but also to evaluate the species of origin. It is in fact well known that a high number (about 14%) of the cultures present both intra and interspecies contaminations, due to the fact that in many laboratories multiple kind of cell cultures may be used, as well as multiple cell lines from the same species. The test suggested by the European Farmacopeia, and used in the Cell Culture Centre to authenticate cell lines, is isoenzyme analysis. The method described in this research could be proposed as an alternative technique to isoenzyme, once evaluated in parallel its reproducibility. The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism analysis described, is based on the use of a pair of primers which anneal to a portion of cytochrome b gene in all the 23 species analyzed. The amplification product is treated with six restriction enzymes and the pattern derived is resolved on agarose gel. For each species, this protocol produced a unique restriction pattern and the origin of the different animal cells tested resulted to be confirmed by this analysis. The sensitivity of the method in detecting inter-species cross-contamination is comparable to that of isoenzyme analysis, but it is less expensive and time consuming and requires a very low amount of DNA. For the other four species, the observed pattern, even if highly reproducible, shows several additional bands; therefore, the species of origin of these four species is confirmed by specific PCR, always performed on mitochondrial DNA target.
Claretta Gioia Losi, Stefania Ferrari, Enrico Sossi, Riccardo Villa, Maura Ferrari. An innovative method for cell line identification and interspecies cross-contaminations: cytochrome b Polymerase Chain Reaction-Restriction Fragment Length Polymorphism analysis, In Vitro Cellular & Developmental Biology-Animal 44:321-329, 2008.