WO2000077176A1

WO2000077176A1 - Method for obtaining low copy transgenes by direct dna transformation

Info

Publication number: WO2000077176A1
Application number: PCT/US2000/016162
Authority: WO
Inventors: Steven L. Spiker; Nandini Mendu; Mara Massel
Original assignee: North Carolina State University
Priority date: 1999-06-14
Filing date: 2000-06-13
Publication date: 2000-12-21
Also published as: AU5484500A

Abstract

A method for the efficient production of a population of cells containing a heterologous DNA of interest at low copy number comprises: providing host cells (e.g., plant cells) for transformation; and then transforming the host cells with a DNA construct comprising, in the 5' to 3' direction, a first matrix attachment region, a DNA of interest, and a second matrix attachment region, wherein the first and second matrix attachment regions are in inverted orientation with respect to one another; where the DNA of interest is incorporated into the host cells at low copy number as compared to that which would occur if the first and second matrix attachment regions were in direct orientation with respect to one another. The DNA of interest may comprise, in the 5' to 3' direction, a transcription initiation region functional in the host cell, and a heterologous DNA segment to be transcribed in the host cell. The transforming step is preferably a direct transformation step, such as electroporation, microprojectile bombardment or the like. Where plant cells are employed, shoots and/or roots, or whole plants, can be regenerated from the transformed cells in accordance with known techniques.

Description

METHOD FOR OBTAINING LOW COPY TRANSGENES BY

DIRECT DNA TRANSFORMATION

Steven L. Spiker, Nandini Mendu, and Mara Massel

Related Applications

This application claims priority from provisional application serial number 60/139,013, filed June 14, 1999, the disclosure of which is incorporated by reference herein in its entirety.

Field of the Invention

The present invention concerns methods for the efficient production of transformed cells containing one or more transgenes in low copy number. In addition, the present invention concerns transformed cells that contain a plurality of different transgenes, each of the different transgenes present in low copy number.

Background of the Invention The use of direct DNA transformation to produce recombinant organisms that contain transgenes in low copy number has been problematic. Direct DNA transformation such as microprojectile bombardment routinely result in incorporation of several (up to hundreds) of copies of the transgene in the host cell genome. In many instances multiple copies of transgenes have been shown to result in the silencing of their expression, interfere with other host genes, and therefore require extensive selection of transformed clones. Approaches to reducing copy number generally involve reducing the efficiency of transformation, but reduced efficiency makes the cell transformation and clonal selection process more time consuming and costly. Present direct DNA transfer procedures result in mostly high copy number transformants that must be identified in order to carry on work with minimal-copy to single-copy transformants. This generates a considerable volume of work and lost efficiency. When it is desired to introduce two or more transgenes into a cell, all at low copy number, the repetition of steps involved can make this goal unrealistic.

Matrix Attachment Regions (MARs; also referred to a scaffold attachment regions or SARs) are genomic DNA sequences that bind specifically to components of the nuclear matrix. See Boulikas, J Cell. Biochem. 52:14 (1993). These sequences are thought to define independent chromatin domains through their attachment to the nuclear matrix. Both transcription and replication are thought to occur at the nuclear matrix. Transformation of a cell using a transgene flanked by one or more MARs has been shown to increase expression of the transgene product, compared to transformation using a construct lacking MARs. See Allen et al., Plant Cell 8:899 (1996); Bonifer et al., EMBO J. 9:2843 (1990); McKnight et al., Proc. Natl. Acad. Sci. USA 89:6943 (1992); Phi-Van et al., Mol. Cell. Biol. 10:2303 (1990)). Flanking a GUS reporter gene with yeast MARs has been reported to result in higher transgene expression in plant cells. Allen et al. Plant Cell 5:603 (1993).

As indicated in U.S. Patent No. 5,773,695 to Thompson, Hall and Spiker at column 4, lines 26-32, MARs have been known to act in an orientation-independent manner (citing Poljak et al., Nucleic Acids Res. 22:4386 (1994)). Thus, in that patent it is taught that the genetic constructs used therein may contain MARs oriented in either direction (5'-3' or 3'-5'), as direct repeats in a single orientation (— —- ), direct repeats in the opposite orientation (<— —), or either of two possible inverted repeats ( ->) or (-»

<-). The goal of Thompson et al. was to increase average expression per gene copy at relatively low copy number (column 2 line 64 to column 3 line 2) rather than to retain transformation efficiency at reduced copy number. The experiments conducted therein involve MARs in direct orientation; the patent teaches that the same result are to be expected regardless of MAR orientation. Thus, the use of MARs would not be expected to provide a solution to the problem of simultaneously achieving efficient transgene transformation at low copy number. Other references concerning MARs include W. Thompson et al, U.S. Patent No.

5,773,689, S. Michalowski and S. Spiker, U.S. Patent Application Serial No. 09/122,400 (filed July 24, 1998), and W. Thompson, U.S. Patent Application Serial No. 09/089,003 (filed June 2, 1998).

Summary of the Invention

A first aspect of the present invention is a method for the efficient production of a population of cells containing a heterologous DNA of interest at low copy number. The method comprises: providing host cells for transformation; and then transforming the host cells with a DNA construct comprising, in the 5' to 3' direction, a first matrix attachment region, a DNA of interest, and a second matrix attachment region, wherein the first and second matrix attachment regions are in inverted orientation with respect to one another; where the DNA of interest is incorporated into the host cells at low copy number as compared to that which would occur if the first and second matrix attachment regions were in direct orientation with respect to one another. The DNA of interest may comprise, in the 5' to 3' direction, a transcription initiation region functional in the host cell, and a heterologous DNA segment to be transcribed in the host cell. The transforming step is preferably a direct transformation step, such as electroporation, microprojectile bombardment or the like. Any cell, including plant or animal cells, can be used as the host cells, but plant cells are preferred. Where plant cells are employed, shoots and/or roots, or whole plants, can be regenerated from the transformed cells in accordance with known techniques. Advantageously, transformed cells can be successively transformed in subsequent transformation steps with different heterologous DNAs containing different DNAs of interest, with each transformation step being performed at high efficiency, to produce transformed cells containing a number of different heterologous DNAs of interest (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), with each of the different DNAs of interest being present at low copy number. For example, in plant cells, it may be desireable to introduce an entire heterologous enzyme pathway in this manner. Thus, a second aspect of the present invention is a transformed host cell containing a plurality (e.g., 3) of different heterologous DNA constructs randomly and stably integrated into the genome of the cell; each of the different DNA constructs comprising, in the 5' to 3' direction, a first matrix attachment region, a DNA of interest, and a second matrix attachment region, wherein the first and second matrix attachment regions are in inverted orientation with respect to one another; wherein each of the different DNA constructs is incorporated into the host cell at low copy number (e.g., a copy number of not more than one, two, three or four).

A third aspect of the present invention is a transformed plant comprising cells containing a plurality of different heterologous DNA constructs randomly and stably integrated into the genome thereof; each of the different DNA constructs comprising, in the 5' to 3' direction, a first matrix attachment region, a DNA of interest, and a second matrix attachment region, wherein the first and second matrix attachment regions are in inverted orientation with respect to one another; wherein each of the different DNA constructs is incorporated into the host cell at low copy number. Plants and plant cells, including both monocots and dicots, are preferred for carrying out the present invention. Animals cells transformed using MARs arranged as inverted repeats were found not to produce transgenes in low copy number. L. Phi- Van, and W. Statling, Dissection of the ability of the chicken lysozyme gene 5' matrix attachment region to stimulate transgene expression and to dampen position effects, Biochemistry 35, 10735-42 (1996).

Brief Description of the Drawings Figure 1 schematically ilustrates the various plasmid vector inserts used to demonstrate the present invention. Figure 2 illustrates the effect of MAR orientation on Gus activity in tobacco cells in culture transformed with the vectors illustrated in Figure 1.

Figure 3 illustratesthe effect of MAR orientation on copy number in a series of tobacco cells transformed with the plasmid vectors of Figure 1. Note the surprisingly low copy number for cell lines containing the inverted MAR vectors.

Detailed Description of the Invention

The term "low copy number" as used herein refers to the incorporation of a heterolgous DNA such as a transgene into a cell at levels less than that which would be obtained under the same transformation conditions, but with the MARs in direct orientation with respect to one another. Low copy number may thus mean 1 , 2, or 3 copies of the particular heterologous DNA per cell. 1 or 2 copies of a given DNA per cell is preferred, and one copy of a given DNA per cell is particularly preferred.

The terms "inverted repeat" and "inverted orientation" as used with respect to MARs herein, refers to a pair of MARs in non-naturally occuring orientation, and where the orientation of one member of the pair of MARs is reversed or flipped as compared to the other (->•«— or < — >). The two MARs in the pair may be the same or different, but are preferably sufficiently homologous to one another so that they hybridize to one another under stringent conditions as defined below.

The terms "plurality of transgenes" and "plurality of DNAs of interest" as used herein with respect to a cell or cells refers to 2 or more different DNAs, each of the different DNAs incorporated into a cell at low copy number as described above. Each transgene is preferably randomly and stably incorporated into the genome of the cell. Thus, such a plurality of DNAs may be 2, 3, 4, 5, 6, 7, 8, 9 or 10 different DNAs of interest incorporated into a cell at low copy number.

The phrase "different DNA constructs" as used herein refers to DNA constructs in which the DNAs of interest inserted between the pair of MARs have a different sequence (i.e., code on expression for different proteins or peptides, RNAs, or the like).

The term "operatively associated," as used herein, refers to DNA sequences on a single DNA molecule which are associated so that the function of one is affected by the other. Thus, a transcription initiation region is operatively associated with a structural gene when it is capable of affecting the expression of that structural gene (i.e., the structural gene is under the transcriptional control of the transcription initiation region). The transcription initiation region is said to be "upstream" from the structural gene, which is in turn said to be "downstream" from the transcription initiation region.

In general, the present invention may be carried out in the manner described in U.S. Patent No. 5,773,689 to Thompson et al., the disclosure of which is incorporated by reference herein in its entirety. Applicants specifically intend that all United States patents and patent applications cited herein be incorporated herein by reference in their entirety. 1. Matrix Attachment Regions.

Any matrix attachment region can be used to carry out the present invention, including MARs of plant, animal, or microbial origin. Examples of suitable MARs are given in U.S. Patent No. 5,773,689 to Thompson et al. and in U.S. Patent No. 5,773,695 to Thompson et al., the disclosures of which are incorporated by reference herein in their entirety. Plant matrix attachment regions are preferred. The RB7 matrix attachment region is preferred, and the tobacco RB7 matrix attachment region is particularly preferred.

Further, in our commonly owned, copending U.S. Patent Application, No. 09/122,400, filed July 24, 1998, the disclosure of which is incorporated by reference herein in its entirety, a number of additional MAR sequences and a MAR motif whose frequency significantly correlates with the binding strength of a MAR, were identified. In the 09/122,400 application, a significant relationship between binding strength and overall AT content was identified. This was the first report of a correlation between the abundance of certain MAR related motifs and MAR binding strength. In addition, the newly identified MAR related motif of local AT rich regions (sections of 20 contiguous nucleotides that are >90% A and/or T), has a higher correlation to MAR binding strength than any of the previously identified motifs. These findings provide a method for the identification of MAR regions in DNA molecules of known nucleotide sequence, useful in identifying MARs for carrying out the present invention. The method comprises identifying, in the known DNA sequence, regions or areas of the sequence which are at least 20 contiguous nucleotides in length and which consist of at least 90% A and/or T nucleotides. The presence of a 20-bp region of > 90% AT indicates a MAR (a single region alone is not sufficient to create a MAR; it takes several); a MAR preferably contain multiple regions (e.g., at least 2, 3, or 4) of > 90% AT. The identification of such regions may be carried out by techniques that are well-known in the art, including sequencing the DNA to be screened and reviewing a printed DNA sequence for such regions. Contiguous fragments of the original DNA sequence that are from one to several kilobases (from about 3,000 nucleotides. 2,000 nucleotides, or about 1,000 nucleotides) in length to about 500, 400. or 300 bases in length, and which encompass the 20-bp regions of > 90% AT can then be isolated (or created de novo by known synthesis techniques) and utilized as MARs. Optionally, the isolated fragments can first be tested for MAR binding strength, for example using an exogenous nuclear matrix binding assay as described therein. The identification of such regions may be carried out by techniques that are well-known in the art, including sequencing the DNA to be screened and reviewing the printed DNA sequence for such regions. Fragments of the original DNA sequence that are from several kilobases in length to about 500, 400, or 300 bases in length, and which encompass the 20-bp regions of > 90% AT can then be isolated (or created de novo by known synthesis techniques) and utilized as MARs. Optionally, the isolated fragments can first be tested for MAR binding strength, for example using an exogenous nuclear matrix binding assay as described therein.

It will be apparent to those of skill in the art that minor sequence variations from the sequences provided above will not affect the function of the MARs of the present invention. MAR DNA sequences include sequences that are functional MARs which hybridize to DNA sequences of other known MARs (or the complementary sequences thereto) under stringent conditions. For example, hybridization of such sequences may be carried out under stringent conditions represented by a wash stringency of 0.3M NaCl, 0.03M sodium citrate, and 0.1% SDS at 60°C, or even 70°C, in a standard in situ hybridization assay. (See J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d ed. 1989)(Cold Spring Harbor Laboratory)). In general, DNA sequences that act as MARs and hybridize to the DNA sequences give above will have at least 70%, 75%, 80%, 85%, 90%, 95% or even 97% or greater sequence similarity to the MAR sequences provided in copending application no. 09/122,400. A particularly preferred MAR for carrying out the present invention is the RB7

MAR, and most particularly the tobacco RB7 MAR, which is disclosed in U.S. Patent No. 5,773,695 to Thompson et al.

2. DNA Constructs. DNA constructs, or "expression cassettes," of the present invention preferably include, 5' to 3' in the direction of transcription, a first matrix attachment region, a transcription initiation region, a DNA of interest such as a structural gene operatively associated with the transcription initiation region, a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal for polyadenylation(e.g., the nos terminator), and a second matrix attachment region, wherein the first and second matrix attachment regions are in inverted orientation. All of these regions should be capable of operating in the cells to be transformed. The termination region may be derived from the same gene as the transcription initiation or promoter region, or may be derived from a different gene.

The transcription initiation region, which preferably includes the RNA polymerase binding site (promoter), may be native to the host organism to be transformed or may be derived from an alternative source, where the region is functional in the host. Other sources include the Agrobacterium T-DNA genes, such as the transcriptional initiation regions for the biosynthesis of nopaline, octapine, mannopine, or other opine transcriptional initiation regions, transcriptional initiation regions from plants, transcriptional initiation regions from viruses (including host specific viruses), or partially or wholly synthetic transcription initiation regions. Transcriptional initiation and termination regions are well known. See, e.g., dGreve, J. Mol. Appl. Genet. 1, 499- 51 1 (1983); Salomon et al, EMBO J. 3, 141-146 (1984); Garfinkel et al., Cell 27, 143- 153 (1983); and Barker et a\., Plant Mol. Biol. 2, 235-350 (1983). The transcriptional initiation regions may, in addition to the RNA polymerase binding site, include regions which regulate transcription, where the regulation involves, for example, chemical or physical repression or induction (e.g., regulation based on metabolites or light) or regulation based on cell differentiation (such as associated with leaves, roots, seed, or the like in plants). Thus, the transcriptional initiation region, or the regulatory portion of such region, is obtained from an appropriate gene which is so regulated. For example, the 1 ,5-ribulose biphosphate carboxylase gene is light-induced and may be used for transcriptional initiation. Other genes are known which are induced by stress, temperature, wounding, pathogen effects, etc.

Structural genes are those portions of genes which comprise a DNA segment coding for a protein, polypeptide, or portion thereof, possibly including a ribosome binding site and or a translational start codon, but lacking a transcription initiation region. The term can also refer to introduced copies of a structural gene where that gene is also naturally found within the cell being transformed. The structural gene may encode a protein not normally found in the cell in which the gene is introduced or in combination with the transcription initiation region to which it is operationally associated, in which case it is termed a heterologous structural gene. Genes which may be operationally associated with a transcription initiation region of the present invention for expression in a plant species may be derived from a chromosomal gene, cDNA, a synthetic gene, or combinations thereof. Any structural gene may be employed. Where plant cells are transformed, the structural gene may encode an enzyme to introduce a desired trait, such as glyphosphate resistance; a protein such as a Bacillus thuringiensis protein (or fragment thereof) to impart insect resistance; or a plant virus protein or fragment thereof to impart virus resistance.

In addition to structural genes, the DNA of interest may encode an antisense oligonucleotide, a ribozyme, a DNA triplex molecule, or any other oligonucleotide species that it is desired to introduce into the target or host cell. The cassette may be provided in a DNA construct which also has at least one replication system. For convenience, it is common to have a replication system functional in Escherichia coli, such as ColEl, pSC101, pACYC184, or the like. In this manner, at each stage after each manipulation, the resulting construct may be cloned, sequenced, and the correctness of the manipulation determined. In addition, or in place of the E. coli replication system, a broad host range replication system may be employed, such as the replication systems of the P-l incompatibility plasmids, e.g., pRK290. In addition to the replication system, there will frequently be at least one marker present, which may be useful in one or more hosts, or different markers for individual hosts. That is, one marker may be employed for selection in a prokaryotic host, while another marker may be employed for selection in a eukaryotic host, particularly a plant host. The markers may be protection against a biocide, such as antibiotics, toxins, heavy metals, or the like; provide complementation, for example by imparting prototrophy to an auxotrophic host; or provide a visible phenotype through the production of a novel compound. Exemplary genes which may be employed include neomycin phosphotransferase (NPTII), hygromycin phosphotransferase (HPT), chloramphenicol acetyltransferase(CAT), nitrilase, and the gentamicin resistance gene. For plant host selection, non-limiting examples of suitable markers are β-glucuronidase, providing indigo production, luciferase, providing visible light production, NPTII, providing kanamycin resistance or G418 resistance, HPT, providing hygromycin resistance, and the mutated aroA gene, providing glyphosate resistance.

The various fragments comprising the various constructs, expression cassettes, markers, and the like may be introduced consecutively by restriction enzyme cleavage of an appropriate replication system, and insertion of the particular construct or fragment into the available site. After ligation and cloning the DNA construct may be isolated for further manipulation. All of these techniques are amply exemplified in the literature and find particular exemplification in Sambrook et al., Molecular Cloning: A Laboratory Manual, (2d Ed. 1989)(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

3. Vectors and Transformation Systems.

Vectors which may be used to transform cells with DNA constructs of the present invention include vectors suitable for direct DNA-mediated transformation such as microparticle bombardment (ballistic or biolistic cell transformation), electroporation, "whisker" techniques, and the like. In general, in one embodiment, the DNA construct comprises a noninfectious,nonintegratingDNA. The DNA construct may optionally be encapsulated in or associated with viral particles, liposomal formulations, charged lipids or the like to facilitate uptake into the cells, as described in U.S. Patent No. 5,459,127. The DNA construct may be simply injected into a tissue such as muscle or skin tissue as described in U.S. Patents Nos. 5,589,466, 5,693,622, and 5,580,859 (the disclosures of all U.S. Patent referenes cited herein are to be incorporated herein by reference).

Microparticles carrying a DNA construct of the present invention, which microparticles are suitable for the ballistic or microprojectile transformation of a cell, are also useful for transforming cells according to the present invention. The microparticle is propelled into a cell to produce a transformed cell. Where the transformed cell is a plant cell, a plant may be regenerated from the transformed cell according to techniques known in the art. Any suitable ballistic cell transformation methodology and apparatus can be used in practicing the present invention. Exemplary apparatus and procedures are disclosed in Stomp et al., U.S. Patent No. 5,122,466; and Sanford and Wolf, U.S. Patent No. 4,945,050 (the disclosures of all U.S. Patent references cited herein are incorporated herein by reference in their entirety). When using ballistic transformation procedures, the expression cassette may be incorporated into a plasmid capable of replicating in the cell to be transformed. Examples of microparticles suitable for use in such systems include 1 to 5 μm gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

Plant species may be transformed with the DNA construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art.

4. Host or Target Cells

The present invention may be used to transform cells, typically eukarytic cells, from a variety of organisms, including animal and plants (i.e., vascular plants), yeast, fungi, etc. As used herein, plants includes both gymnosperms and angiosperms (i.e., monocots and dicots). As used herein, animals includes mammals (e.g., dog, cat, pig, rat, mouse, rabbit, sheep), including both primate and non-primate mammals, and "animals" including avian species such as chicken and turkey. Transformation according to the present invention may be used to increase expression levels of transgenes in stably transformed cells. Cells may be transformed while in cell culture; while in vivo or in situ in a tissue, organ, or in an intact organism, depending upon the technique most suitable for the particular organism involved.

Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector of the present invention. The term "organogenesis," as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers; the term "embryogenesis," as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

Plants of the present invention may take a variety of forms. The plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the expression cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species). The transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. A dominant selectable marker (such as npt II) can be associated with the expression cassette to assist in breeding. Seed can be generated on and collected from such plants in accordance with known techniques to produce seed capable of regenerating plants of the invention.

Note that, when a subsequent transformation step is carried out on cells that have been previously transformed in accordance with the present invention (e.g., the transformation of first-generation transformed cells), the subsequent transformation step may be performed on the same cells previously transformed or on progeny thereof. Indeed, various steps such as culturing, regeneration of a tissue or organism, etc., may optionally intervene between a prior transformation step and a subsequent transformation step.

Plants which may be employed in practicing the present invention include (but are not limited to) canola, sorghum, tobacco (Nicotiana tabacum), potato (Solanum tuberosum), soybean (glycine max), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot escidenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Fiats casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolid), almond (Prunus amygdalus), sugar beets (Beta vulgaris), corn (Zea mays), wheat, oats, rye, barley, rice, vegetables, ornamentals, and conifers. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Pisum spp.) and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea. (Rhododendron spp.), hydrangea (Mαcrophyllα hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (T lipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (dianthus caryophyllus), poinsettia (Euphorbia pulcherima), and chrysanthemum. Gymnosperms which may be employed to carrying out the present invention include conifers, including pines such as loblolly pine (Pinus taedά), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contortd), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glaucά); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis); Eucalyptus and Polonia.

The methods of the invention are preferably carried out under low osmotic pressure conditions. By "low osmotic pressure" is meant an osmotic pressure less than that of 100 or 200 milliMolar of sucrose, or the osmotic equivalent thereof. For example, The culture medium in which transformation is carried out preferably contains less than 100 or 200 milliMolar, combined, of manitol, sorbitol, and/or sucrose.

The examples which follow are set forth to illustrate the present invention, and are not to be construed as limiting thereof.

EXAMPLE 1

Effect of Inverted repeats in Tobacco NT-1 Cells

The methods of the present invention were carried out in tobacco NT-1 cells in culture. Procedures were in accordance with known techniques as described in G. Allen et al., The Plant Cell 8, 899-913 (1996). The cell lines were transformed with the constructs shown in Figure 1. All cells received the selection plasmid with an insert containing the NPTII gene conferring kanamycin resistance. Cells also received one of the following reporter plasmids: The reporter gene (GUS) alone, the reporter gene flanked with the' RB7 MAR as direct repeats (indicated by arrows of the same direction) or either of two possible inverted repeats (indicated by arrows of opposite directions). As shown in Figure 2. cell lines containing MARs as either direct or inverted repeats have on average a much higher level of specific activity of the reporter gene product, GUS, as compared to the No MAR lines. Orientation of MARs in Figure 2 is indicated by arrows as in Figure 1. In Figure 3, the effect of MAR orientation on copy number is shown. The cell lines in which no MAR flanked the reporter gene or in which MARs as direct repeats flanked the reporter gene were mostly high copy lines — up to 600 and 200 copies per genome for direct repeat MARs and no MARs respectively. Nearly all the cell lines in which the flanking MARs were oriented as inverted repeats contained a single copy of the transgene as determined by quantitative PCR and Southern hybridization.

EXAMPLE 2 Effect of High Osmotic Conditions

The foregoing experiment was repeated, except that the culture medium was changed to include 125 milliMolar of manitol and 125 milliMolar of sorbitol. In this experiment, only 5 of 30 transformed cell lines contained single or low copies of the reporter gene (GUS) when one of the two possible inverted repeats was investigated. There was a marked consistency in most of the rest of the transformants at around 70 + 10 copies

The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is described by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A method for the efficient production of a population of cells containing a heterologous DNA of interest at low copy number, said method comprising: providing host cells for transformation; and then transforming said host cells with a DNA construct comprising, in the 5 ' to 3' direction, a first matrix attachment region, a DNA of interest, and a second matrix attachment region, wherein said first and second matrix attachment regions are in inverted orientation with respect to one another; where said DNA of interest is incorporated into said host cells at low copy number as compared to that which would occur if said first and second matrix attachment regions were in direct orientation with respect to one another.

2. A method according to claim 1, wherein said DNA of interest comprises, in the 5 ' to 3 ' direction, a transcription initiation region functional in said host cell, and a heterologous DNA segment to be transcribed in said host cell.

3. A method according to claim 1, wherein said transforming step is a direct transformation step.

4. A method according to claim 1, wherein said transforming step is a microprojectile bombardment step.

5. A method according to claim 1, wherein said DNA construct comprises a noninfectious, nonintegrating DNA.

6. A method according to claim 1, wherein said host cells are animal cells.

7. A method according to claim 1, wherein said host cells are plant cells.

8. A method according to claim 7, further comprising the step of regenerating shoots from said transformed plant cells.

9. A method according to claim 7, further comprising the step of regenerating roots from said transformed plant cells.

10. A method according to claim 7, further comprising the step of regenrating a plant from said transformed plant cells.

11. A method according to claim 1 , wherein said low copy number is not more than three.

12. A method according to claim 1, wherein said low copy number is not more than one.

13. A method for the efficient production of a cell containing a plurality of different heterologous DNAs of interest at low copy number, said method comprising:

(a) providing a host cell for transformation; and then (b) transforming said host cell with a first DNA construct comprising, in the 5' to 3' direction, a first matrix attachment region, a DNA of interest, and a second matrix attachment region, wherein said first and second matrix attachment regions are in inverted orientation with respect to one another; where said first DNA of interest is incorporated into said host cell at low copy number as compared to that which would occur if said first and second matrix attachment regions were in direct orientation with respect to one another; to produce a first generation transformed host cell; and then

(c) transforming said first generation host cell with a second DNA construct comprising, in the 5 ' to 3 ' direction, a first matrix attachment region, a second

DNA of interest, and a second matrix attachment region, wherein said first and second matrix attachment regions are in inverted orientation with respect to one another; where said second DNA of interest is incorporated into said host cell at low copy number as compared to that which would occur if said first and second matrix attachment regions were in direct orientation with respect to one another; to produce a second generation transformed host cell containing two different heterologous DNAs of interest, each at low copy number.

14. A method according to claim 13, wherein said first and second

DNA of interest comprises, in the 5' to 3' direction, a transcription initiation region functional in said host cell, and a heterologous DNA segment to be transcribed in said host cell.

15. A method according to claim 13, wherein said transforming steps are direct transformation steps.

16. A method according to claim 13, wherein said transforming steps are microprojectile bombardment steps.

17. A method according to claim 13, wherein said DNA construct comprises a noninfectious, nonintegrating DNA.

18. A method according to claim 13, wherein said host cell is an animal cell.

19. A method according to claim 13, wherein said host cell is a plant cell.

20. A method according to claim 19, further comprising the step of regenerating shoots from said transformed plant cell.

21. A method according to claim 19, further comprising the step of regenerating roots from said transformed plant cell.

22. A method according to claim 19, further comprising the step of regenrating a plant from said transformed plant cells.

23. A transformed host cell containing one or more different heterologous DNA constructs in low copy number, each of said heterologous DNA constructs comprising, in the 5' to 3' direction, a first matrix attachment region, a DNA of interest, and a second matrix attachment region, wherein said first and second matrix attachment regions are in inverted orientation with respect to one another.

24. A plant comprising transformed host cells according to claim 23.

25. A transformed host cell containing a plurality of different heterologous DNA constructs randomly and stably integrated into the genome of said cell; each of said different DNA constructs comprising, in the 5' to 3' direction, a first matrix attachment region, a DNA of interest, and a second matrix attachment region, wherein said first and second matrix attachment regions are in inverted orientation with respect to one another; wherein each of said different DNA constructs is incorporated into said host cell at low copy number.

26. A transformed host cell according to claim 25 containing 3 different

DNA constructs.

27. A transformed host cell according to claim 26, each of said different DNA constructs containing a different DNA of interest.

28. A transformed host cell according to claim 25 wherein each of said different DNA constructs is incorporated into said host cell at a copy number of 1.

29. A transformed host cell according to claim 25, wherein said host cell is an animal cell.

30. A transformed host cell according to claim 25, wherein said host cell is a plant cell.

31. A transformed plant comprising cells containing a plurality of different heterologous DNA constructs randomly and stably integrated into the genome thereof; each of said different DNA constructs comprising, in the 5' to 3' direction, a first matrix attachment region, a DNA of interest, and a second matrix attachment region, wherein said first and second matrix attachment regions are in inverted orientation with respect to one another; wherein each of said different DNA constructs is incorporated into said host cell at low copy number.

32. A transformed plant according to claim 31, said cells containing 3 different DNAs of interest.

33. A transformed plant according to claim 31 wherein each of said different DNA constructs is incorporated into said cells at a copy number of 1.

34. A transformed plant according to claim 31 , wherein said plant is a monocot.

35. A transformed plant according to claim 31 , wherein said plant is a dicot.

36. Plant seed that regenerates into a plant of claim 31.