Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...
Reexamination Certificate
1999-10-07
2002-05-21
Guzo, David (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C435S320100, C435S252300, C435S252330, C435S469000, C435S468000, C435S410000, C435S411000, C435S412000, C435S414000, C435S415000, C435S417000, C435S430000, C435S470000, C536S023100, C536S023200, C536S023600, C536S024100, C800S278000, C800S280000, C800S293000, C800S295000, C800S298000, C800S312000, C800S317200, C800S317300, C800S317400, C800S320100, C800S320200, C800S320300
Reexamination Certificate
active
06392121
ABSTRACT:
TECHNICAL FIELD
The present invention is related to genetic engineering of plants. The invention is particularly related to the transformation of plants using recombinant DNA techniques to amplify a gene of interest and express a protein of interest.
BACKGROUND OF THE INVENTION
A predominant mode of plant transformation employs
A. tumefaciens,
in which a transforming DNA (T-DNA) is modified to incorporate a desired foreign gene. The recombinant T-DNA contains the desired foreign gene between flanking non-coding regulatory sequences and the left and right border regions of the wild-type tumor-inducing (Ti) plasmid. The recombinant T-DNA can be provided as part of an integrative plasmid, which integrates into a wild-type Ti plasmid by homologous recombination. Typically, however, the recombinant T-DNA is provided in a binary vector and transferred into a plant cell through the action of trans-acting vir genes on a helper Ti plasmid. The T-DNA integrates randomly into the nuclear genome with some of the transformants permitting expression of the desired protein (Zambryski, 1988). Because of the random integration event of the T-DNA into the nuclear chromosome, variability of transcription level is expected, and transformants are screened to identify those showing the highest levels of foreign gene expression. Those transformants expressing the highest levels of foreign protein can be propagated and multiplied in tissue culture before transplanting to soil.
Many plant species previously recalcitrant to gene transfer are now amenable, including cereal crops (McElroy et al., 1994). Plants have the capacity to express foreign genes from a wide range of sources, including viral, bacterial, fungal, insect, animal, and other plant species. In single-copy nuclear transgenics, foreign protein in excess of 1% of total protein is often achieved (Hiatt et al., 1989). Further, assembly and processing of complex animal proteins in plants is possible, e.g., human serum albumin (Sijmons et al., 1990) and secretory antibodies (Ma et al., 1995a). Recently, expression of correctly processed avidin was reported in corn seed at a level of 2% of the total soluble protein (Hood et al., 1997). It has been estimated that the cost of recombinant protein production in plants (assuming the foreign protein is 10% of total protein) can be 10 to 50 times less than in
E. coli
by fermentation (Kusnadi et al., 1997).
Plants have been used as expression systems for vaccine antigens (Mason et al., 1995). The expression of vaccine antigens in tobacco plants has been reported and the plant material has been shown to be orally immunogenic in mice (work reviewed by Mason et al., 1995; Arntzen et al., 1996). Complex antibodies have also been expressed in plants, which correctly processed and assembled the antibody chains into IgG and secretory IgA forms (review by Ma et al., 1995b). In the latter case, four different genes were coordinately expressed, including the IgA heavy and light chains, the joining component, and the secretory component, which faithfully assembled in plant cells. Further, expression and accumulation of antibodies in corn and soybean seeds has been reported.
However, a major limitation in the use of plants for expression and delivery of a protein of interest is the rather low level of expression usually obtained, which ranges from 0.01% to 2% of the total soluble protein. For example, soybeans contain 40% protein by weight, yet current methods for foreign protein expression yield no more than 2% of the total protein in seeds. Synthetically produced recombinant vaccine proteins, which avoid the hazards associated with using live or attenuated virus, can be produced in cell culture systems, e.g., hepatitis B surface antigen (Cregg et al., 1987) and dengue virus proteins (Sugrue et al., 1997). However, the cost of cell culture systems is often so high as to preclude vaccination on a large scale, particularly in poor countries.
In applications requiring overexpression of a purified protein of interest, high-level expression would greatly facilitate the purification process. Therefore, a method of amplifying a gene of interest and overproducing a protein of interest in recombinant plants is desired.
Previous techniques are, however, inherently self-limiting by virtue of “successful” transformation affording only one or a few functional copies of the foreign gene integrated into the plant genome. Efforts to increase the level of expression under such circumstances are therefore limited to optimizing the promoter and/or enhancer sequences, using synthetic versions of the foreign gene optimized for expression in the plant host, optimizing the termination sequence, optimizing expression of transcription factors, and the like. These measures can be expected to enhance expression of the desired antigen, although such enhancement is still limited by the copy number of the foreign gene. True “amplification” of the foreign gene in plant cells, in which multiple functional copies of the gene are generated either extrachromosomally or integrated into the plant chromosome, is desired if much greater levels of protein expression are to be achieved. The geminiviruses are interesting candidates for producing marked amplification of transgenes in plants.
Members of the plant virus taxonomic family Geminiviridae are unique among viruses in possessing twinned or geminate virions. They are also unusual among plant viruses in that they possess single-stranded circular DNA genomes. The three genera of Geminiviridae are: the leafhopper-transmitted Mastreviruses (type member: maize streak virus, MSV); the leaf- and planthopper-transmitted Curtoviruses (type member: beet curly top virus, BCTV); and the whitefly-transmitted Begomoviruses (type member: bean golden mosaic virus, BGMV). Until recently, the three genera were known as Subgroups I, II and III, respectively. Mastreviruses and Curtoviruses have only a single genomic component of approximately 2.5 to 2.8 kb; Begomoviruses may have one or two components of the same size, one of which is dependent on the other for replication. Mastreviruses have the simplest organization, with Curtoviruses and Begomoviruses sharing a very similar and more complex organization. An overview of the genetic organization of geminivirus genomes is shown in FIG.
1
.
The geminiviruses replicate via a rolling circle mechanism, analogous to that used by phage &PHgr;X174 and ssDNA plasmids of gram positive microorganisms. The only exogenous a protein required for replication is the viral replication initiation (Rep) protein encoded by a geminiviral replicase gene. This multifunctional protein initiates replication at a conserved stem loop structure found in the viral origin of replication by inducing a nick within a conserved nonanucleotide motif (TAATATTA↓C) found in the intergenic loop sequence. Transcription of the viral genome is bidirectional with transcription initially within the intergenic (IR) region. Rep also has functions involved in controlling the plant cell cycle, and possibly also in modulating the expression of host genes involved in DNA replication (reviewed by Palmer et al., 1997b). The Rep protein can act in trans, that is, it need not be expressed by the viral replicon itself, but can be supplied from another extrachromosomal viral replicon, or even from a nuclear transgene (Hanley-Bowdoin et al., 1990). The cis requirements for viral replication are the viral intergenic region/s (IR), which contain sequences essential for initiation of rolling circle replication (the long intergenic region (LIR) of Mastreviruses, or the intergenic region of other geminiviruses) and synthesis of the complementary strand (the short IR (SIR) of Mastreviruses).
Infectious clones of geminiviruses are commonly constructed as tandem dimers or partial dimers of the virus genome, usually with the origin of replication sequences duplicated. This facilitates escape of the cloned virus from the cloning vector sequences by a replicative release mechanism mediated by the Rep protein inducing a nick
Arntzen Charles
Hefferon Kathleen L.
Mason Hugh S.
Mor Tsafrir S.
Palmer Kenneth E.
Boyce Thompson Institute for Plant Research
Guzo David
Palmer & Dodge LLP
Williams Kathleen M.
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