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ABI Research Summaries - Vibha Srivastava, Ph.D. Dr. Srivastava's research objectives are (i) to develop molecular tools for precise integration of foreign genes in the plant genome, (ii) to develop strategies for generating environmentally safe transgenic plants. Genetically modified (GM) plants are very important for the growth of agriculture. Majority of the GM plants produced by the conventional techniques are not suitable for long-term propagation because they frequently undergo gene silencing. The position and the complexity of the gene integration locus are the major factors that contribute to the gene silencing phenomenon. Therefore, to prevent gene silencing, it is important to control the location and the pattern of the integration of the foreign gene. My laboratory is developing DNA recombination based strategies for obtaining the integration of a single copy of the foreign gene in a pre-determined genomic location. Although, the primary goal of our research is to improve crop genetic engineering methods, the strategies we have developed are also suitable for deciphering function of novel genes. Further, to address the concerns about the spread of transgenes from GM crops to the wild species, we are testing a molecular strategy for gene containment in GM plants. Site-specific gene integration in plants (i) Recombinase-mediated gene targeting Using P1 phage site-specific recombination system, Cre-lox, we have developed a strategy for integrating a single copy of the foreign gene in a designated genomic location. This strategy works with direct DNA transformation methods. Currently we are studying the efficiency of the protocol and the stability of the site-specifically integrated gene. The procedure allows the selection for site-specific integration events but does not prevent random integrations. Therefore, we are developing a negative selection strategy that will allow us to de-select random integrations. Further, we would like to make this procedure suitable for any plant transformation method. To address that, we are working on a strategy that will allow us to obtain site-specific gene integration by crossing in two transgenic alleles. (ii) Homologous recombination (HR) mediated gene targeting. Homologous recombination is a basic cellular process that can be utilized to target a foreign DNA into the homologous genomic location. However, in higher plants HR-mediated gene targeting works extremely inefficiently. It is clear that culture cells, which divide mitotically, undergo non-homologous recombination much more efficiently. Efficient HR occurs in the meiotic cells. Since it is not possible to culture meiotic cells in vitro, we have developed a molecular tool to deliver DNA in meiotic cells of a plant. Currently, we are testing if efficient HR-based gene targeting can be obtained by introducing DNA in the meiotic cells. This technique will not only be useful for genetic engineering of crops but also be valuable for deciphering the functions of novel genes. Transgene containment Field grown GM plants pose the threat of transferring transgenes to wild species by cross-fertilization. This transgene escape may have negative impact on the environment. GM plants contain transgene in both target and non-target tissues. Majority of GM plants require that the transgene be expressed only in the few different types of tissue and that the transgene be transmitted to the next generation. Since there is no feasible method of introducing DNA tissue-specifically, we propose to delete the introduced DNA tissue-specifically. To address this issue, we have designed a Cre-lox based method to delete transgene from pollens. GM plants produced with this method will make transgene-free pollen and therefore will not pose the threat of spreading the transgene in the environment. Selected Publications: Srivastava V and Ow DW Rare instances of Cre-mediated deletion product maintained in transgenic wheat. Plant Mol. Biol. (in press) Srivastava V and Ow DW. Site-specific gene integration in rice. Mol. Breed. 8: 345-350, 2001. Srivastava V and Ow DW Single copy primary transformants of maize obtained through the co-introduction of a recombinase-expressing construct. Plant Mol. Biol. 46: 561-566, 2001. Srivastava V, Anderson O D, Ow DW Single-copy transgenic wheat generated through the resolution of complex integration patterns. Proc. Natl. Acad. Sci. (USA) 96:11117-11121, 1999. Altpeter A, Vasil V, Srivastava V, Vasil IK Integration and expression of the high molecular weight glutenin subunit 1Ax1 gene into wheat. Nat. Biotechnol. 14: 1155-1159, 1996. Srivastava V, Vasil V, Vasil IK Molecular characterization of the fate of transgenes in transformed wheat. Theor. Appl. Genet. 92: 1031-1037, 1996. Vasil V, Srivastava V, Castillo AM, Vasil IK Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos. Biotechnol. 11:1553-1558, 1993. Contact Information Vibha Srivastava, Ph.D. 115 Plant Science bldg. Phone: (479) 575-4872 |