Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1998-06-02
2001-05-29
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C260S66500B, C260S66500B, C260S66500B, C435S468000, C435S469000, C536S024100
Reexamination Certificate
active
06239328
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for reducing the variability of expression, increasing expression, and/or reducing gene silencing in transgenic plants.
BACKGROUND OF THE INVENTION
Agricultural biotechnology, and particularly plant biotechnology, has become recognized as one of the principal areas for the application of biotechnology techniques. Systems exist for transforming plant cells and regenerating complete plants from the transformed cells; structural gene and gene regulatory regions continue to be identified; and the need for plants with genetically engineered traits such as insect resistance and drought resistance remains strong.
A problem with the expression of foreign genes in plants is the clonal variation in the expression of the same gene in independent transformants: a problem referred to as “position effect” variation. No completely satisfactory method of obviating this problem has yet been developed, and there is accordingly a continued need for solutions to this problem.
L. Mlynarova,
Approaching the Lower Limits of Transgene Variability
, The Plant Cell 8, 1589-1599 (1996) is concerned with Tobacco only. Monocots are neither nor disclosed nor suggested. In addition, Mlynarova is only concerned with an F2 generation produced by selfing the F1 generation.
PCT Application WO94/07902, titled Method for Increasing Expression and Reducing Expression Variability of Foreign Genes in Plant Cells, (published 14 Apr. 1994) it is stated that “where two matrix attachment regions are employed, they may be the same or different.” (page 5, lines 13-15).
SUMMARY OF THE INVENTION
A method of reducing gene silencing, increasing expression, and/or reducing expression variability of foreign DNA in plants or plant cells comprises providing a plant cell capable of regeneration; and then transforming said plant cell with a DNA construct comprising an expression cassette, which construct comprises, in the 5′ to 3′ direction, a first matrix attachment region, a transcription initiation region, a structural gene positioned downstream from said transcription initiation region and operatively associated therewith, and a second matrix attachment region, wherein said first and second matrix attachment regions are different.
In a preferred embodiment of the foregoing, a method of making recombinant plants with reduced silencing of expression, increased expression, and/or reduced expression variability of of foreign genes therein comprises: (a) providing a plant cell, preferably a monocot plant cell, capable of regeneration; (b) transforming the plant cell with a DNA construct comprising an expression cassette, which construct comprises, in the 5′ to 3′ direction, a transcription initiation region, a structural gene positioned downstream from the transcription initiation region and operatively associated therewith, a first matrix attachment region positioned 5′ to the structural gene, and a second matrix attachment region positioned 3′ to the structural gene, to produce a transformed plant cell; then (c) propagating a plant from said transformed plant cell to provide a first transgenic plant; and then (d) sexually propagating said first transgenic plant to provide a subsequent transgenic plant having reduced silencing of expression of foreign genes therein. In a preferred embodiment, the plant is a monocot, and the subsequent transgenic plant is a hybrid of one parent that contains the aforesaid expression cassette and another that does not.
A second aspect of the present invention is a DNA construct comprising an expression cassette, which construct comprises, in the 5′ to 3′ direction, a transcription initiation region, a structural gene positioned downstream from the transcription initiation region and operatively associated therewith, a first AR positioned: 5′ to the transcription initiation region and a second MAR positioned 3′ to the structural gene, where the first and second MAR are different from one another.
A third aspect of the present invention is a DNA construct as given above carried by a plant transformation vector.
A fourth aspect of the present invention is a plant cell containing a DNA construct as given above.
A fifth aspect of the present invention is a recombinant plant comprising transformed plant cells, the transformed plant cells containing a heterologous DNA construct as given above.
REFERENCES:
patent: 5773689 (1998-06-01), Thompson et al.
patent: 5773695 (1998-06-01), Thompson et al.
patent: 94/07902 (1994-04-01), None
Phi-Van et al. Mol. Cell. Biol. 10(5): 2302-2307, May 1990.*
Stief et al. Nature 341: 343-345, Sep. 1989.*
Breyne et al.; Characterization of a Plant Scaffold Attachment Region in a DNA Fragment That Normalizes Transgene Expression in Tobacco,The Plant Cell, 4:463-471 (1992).
Clapham et al.; Enhancement of Short- and Medium-term Expression of Transgenes in Embryogenic Suspensions ofPicea Abies(L.) Karst,Journal of Experimental Botany, 46(287):665-662 (1995).
Allen et al.; High-Level Transgene Expression in Plant Cells: Effects of a Strong Scaffold Attachment Region from Tobacco,The Plant Cell, 8:899-913 (1996).
Han et al.; Martrix Attachment Regions (MARs) Enhance Transformation Frequency and Transgene Expression in Poplar,Transgenic Research, 6:415-420 (1997).
Allen et al.; Scaffold Attachment Regions (SARS) Increase Reporter Gene Expression in Stably Transformed Plant Cells,Abstract #560, Supplement to Plant Physiology, 102(1):100 (May 1993).
Allen et al.; Scaffold Attachment Regions Increase Reporter Gene Expression in Stably Transformed Plant Cells,The Plant Cell, 5:603-613 (1993).
Liu et al.; The Influences of Two Plant Nuclear Matrix Attachment Regions (MARs) on Gene Expression in Transgenic Plants,Plant Cell Physiol., 39(1):115-123 (Jan. 1998).
Fox David T.
Myers Bigel & Sibley & Sajovec
North Carolina State University
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