Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-12-20
2002-11-26
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S069800, C435S320100, C435S419000, C536S023600, C800S287000, C800S298000, C800S306000, C800S320100
Reexamination Certificate
active
06486382
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of plant transformation in which a DNA construct carrying a gene encoding the green fluorescent protein (GFP) is introduced into plant cells which are then screened for the presence of the protein and transformed cells are regenerated into transgenic plants. In particular, the present invention provides methods for circumventing the cellular toxicity of the GFP by regulating expression of the gene encoding the protein or directing the protein to a subcellular compartment where it is not toxic to the cell. The present invention provides DNA constructs for cell transformation in which expression of a gene encoding the GFP is placed under the control of an inducible, constitutive or tissue-specific promoter. In addition, DNA constructs are provided in which a nucleotide sequence encoding the GFP is operably linked to a signal or targeting sequence which directs the expressed protein to a subcellular compartment where the protein is not toxic to the cell. Moreover, the present invention provides a nucleotide sequence encoding GFP that is optimized for expression of the GFP gene in plants and to GFP-encoding nucleotide sequences that code for light-shifted versions of GFP. The present invention also provides a method for selecting plant cells transformed with a gene encoding a screenable marker flanked on the 5-prime and 3-prime ends with a recombinase-specific target sequence, and introducing a gene encoding a site specific recombinase into the transformed plant cells and selecting transformed plant cells that no longer express the screenable marker. In addition, the present invention provides a method of reducing GFP toxicity by transforming plant cells with a gene encoding the GFP together with a gene encoding an oxygen scavenger such as superoxidase dismutase.
2. Background
Expression vectors include at least one genetic marker that allows transformed cells to be either recovered by negative selection, i.e. inhibiting growth of cells that do not contain the selectable marker gene, or by screening for product encoded by the genetic marker. Many of the commonly used selectable marker genes for plant transformation were isolated from bacteria and code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide. Other selectable marker genes encode an altered target which is insensitive to the inhibitor.
The most commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene, isolated from Tn5, which when placed under the control of plant regulatory signals confers resistance to kanamycin. Fraley et al.,
Proc. Natl. Acad. Sci. U.S.A.,
80: 4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al.,
Plant Mol. Biol.,
5: 299 (1985).
Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase, the bleomycin resistance determinant. Hayford et al.,
Plant Physiol.
86: 1216 (1988), Jones et al.,
Mol. Gen. Genet.,
210: 86 (1987), Svab et al.,
Plant Mol. Biol.
14: 197 (1990), Hille et al.,
Plant Mol. Biol.
7: 171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or broxynil. Comai et al.,
Nature
317: 741-744 (1985), Gordon-Kamm et al.,
Plant Cell
2: 603-618 (1990) and Stalker et al.,
Science
242: 419-423 (1988).
Other selectable marker genes for plant transformation are not of bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al.,
Somatic Cell Mol. Genet.
13: 67 (1987), Shah et al.,
Science
233: 478 (1986), Charest et al.,
Plant Cell Rep.
8: 643 (1990).
Although many of these markers have been used for selecting transformed plant tissue, these selection systems involving toxic chemical agents can have disadvantages or limitations. One disadvantage is that it may be difficult to recover normal, viable transformed plants directly from chemical selection. Everett et al.,
Bio/Technology
5: 1201-1204 (1987). Another disadvantage is that not all selectable marker systems work for all tissues, in all plant species, due in part to differences in sensitivity of a particular tissue or plant species to the selective agent. The success of any given marker for transformation of a given plant species is not easily predicted. Moreover, potential regulatory issues surrounding the use of antibiotic resistance genes and the use of herbicide resistance genes for plant species capable of outcrossing with weedy species are additional disadvantages of these markers.
Another class of marker genes for plant transformation require screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include &bgr;-glucuronidase (GUS), &bgr;-galactosidase, luciferase, and chloramphenicol acetyltransferase Jefferson, R. A.,
Plant Mol. Biol. Rep.
5: 387 (1987)., Teeri et al.,
EMBO J.
8: 343 (1989), Koncz et al.,
Proc. Natl. Acad. Sci. U.S.A.
84: 131 (1987), De Block et al.,
EMBO J.
3: 1681 (1984). Another approach to the identification of relatively rare transformation events has been use of a gene that encodes a dominant constitutive regulator of the
Zea mays
anthocyanin pigmentation pathway. Ludwig et al.,
Science
247: 449 (1990).
Although chemical selection of plant cells transformed with selectable marker genes has been successful with plant species and varieties that are easily cultured in vitro, the choice of selectable marker systems that have been shown to be successful for cereals and many other agronomically important plant species is very limited. In general, plant species that tend toward organogenesis and/or shoot propagation have been difficult to transform by means of chemical selection. The success rate with these plant species continues to improve, however, as evidenced by recent advances in Type I selection of maize inbreds and small grain cereals such as barley. Koziel et al.,
Bio/Technology
11: 194-200 (1993) and Mendel et al. In:
Transformation of Plants and Soil Microorganisms,
Wang et al. eds., Cambridge Press (1995).
Likewise, there has been little success in using visual screening methods for primary identification of transformed cells. The GUS gene was used to investigate germline transmission. McCabe et al.,
Plant Physiol.,
87(3): 671 (1988) and McCabe et al.,
Plant Cell Tissue Organ Cult.
33 (3): 227 (1993). Histochemical staining for GUS activity was used to locate transgenic sectors in cotton and soybean transformants that ultimately produced transgenic seeds. Since histochemical analysis for GUS activity requires destruction of portions of the presumptively transformed plant tissue, this method is labor intensive and impractical for routine production of transgenic plants. This method is particularly unsuitable for plant species such as maize and other cereals in which transformants are recovered, even under optimum conditions, at low frequency. Recovery of transformed progeny was reported once in barley using GUS expression as a screening tool, but the method was found to be very labor-intensive. Ritala et al.,
Plant Mol. Biol.
24: 317-325 (1994). There have been no reports at all of success with GUS or other screenable markers with maize.
More recently, in vivo methods for vis
Bidney Dennis
Bowen Benjamin
Gordan-Kamm William
Miller Michael D.
Pierce Dorothy A.
Mehta Ashwin
Nelson Amy J.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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