Directed excision of a transgene from the plant genome

SummaryThe effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, β-glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALSr), were integrated into the genome of tobacco and Arabidopsis. The ALSr gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALST gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The β-glucuronidase gene remained active. The excision efficiency varied in F1 progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox × Cre F1 progeny were chimeric and some F2 progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.

[1]  Jonathan D. G. Jones,et al.  Relative strengths of the 35S califlower mosaic virus, 1′, 2′, and nopaline synthase promoters in transformed tobacco sugarbeet and oilseed rape callus tissue , 1988, Molecular and General Genetics MGG.

[2]  R. Malmberg.  Molecular biology of plants , 1985 .

[3]  R. Hoess,et al.  Bacteriophage P 1 Site-specific Recombination , 2001 .

[4]  Kathleen Y. Lee,et al.  A chimaeric hygromycin resistance gene as a selectable marker in plant cells , 1985, Plant Molecular Biology.

[5]  H. V. Gelder.  The Netherlands , 2004, Constitutions of Europe (2 vols.).

[6]  D. Ow,et al.  Intra- and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase. , 1990, Gene.

[7]  B. Mcclintock,et al.  Chromosome organization and genic expression. , 1951, Cold Spring Harbor symposia on quantitative biology.

[8]  N. Sternberg,et al.  Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. , 1981, Journal of molecular biology.

[9]  J. K. Smith,et al.  Isolation and characterization of plant genes coding for acetolactate synthase, the target enzyme for two classes of herbicides. , 1987, Plant physiology.

[10]  P. Caimi,et al.  Site-directed recombination in the genome of transgenic tobacco , 1990, Molecular and General Genetics MGG.

[11]  N. Fedoroff,et al.  Mobility of the maize suppressor-mutator element in transgenic tobacco cells. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Van Montagu,et al.  Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[13]  R. Hoess,et al.  Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein. , 1984, The Journal of biological chemistry.

[14]  R. Hoess,et al.  The role of the loxP spacer region in P1 site-specific recombination. , 1986, Nucleic acids research.

[15]  P. Starlinger,et al.  Detection and abundance of mRNA and protein encoded by transposable element Activator (Ac) in maize , 1991, Molecular and General Genetics MGG.

[16]  Kathleen Y. Lee,et al.  The molecular basis of sulfonylurea herbicide resistance in tobacco , 1988, The EMBO journal.

[17]  M. Matzke,et al.  Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences , 1991, Plant Molecular Biology.

[18]  J. Mol,et al.  Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. , 1990, The Plant cell.

[19]  R. Hehl,et al.  Properties of the maize transposable element Activator in transgenic tobacco plants: a versatile inter-species genetic tool. , 1990, The Plant cell.

[20]  R. Hoess,et al.  Studies on the properties of P1 site-specific recombination: Evidence for topologically unlinked products following recombination , 1983, Cell.

[21]  R. Larossa,et al.  Toxic accumulation of alpha-ketobutyrate caused by inhibition of the branched-chain amino acid biosynthetic enzyme acetolactate synthase in Salmonella typhimurium , 1987, Journal of bacteriology.

[22]  E. Finnegan,et al.  The maize transposable element Ac excises in progeny of transformed tobacco , 1989, Plant Molecular Biology.

[23]  D. Ow,et al.  Gene transfer with subsequent removal of the selection gene from the host genome. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[24]  S. Rothstein,et al.  Transformation of a partial nopaline synthase gene into tobacco suppresses the expression of a resident wild-type gene. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[25]  D. Haas,et al.  Genetic and molecular characterization of the Pseudomonas plasmid pVS1. , 1984, Plasmid.

[26]  C. Napoli,et al.  Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. , 1990, The Plant cell.

[27]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[28]  S M Burgess,et al.  beta-Glucuronidase from Escherichia coli as a gene-fusion marker. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[29]  E. Dennis,et al.  Transposable elements can be used to study cell lineages in transgenic plants. , 1989, The Plant cell.

[30]  Jonathan D. G. Jones,et al.  Visual Detection of Transposition of the Maize Element Activator (Ac) in Tobacco Seedlings , 1989, Science.