Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Via agrobacterium
Reexamination Certificate
2000-11-22
2004-02-03
Horlick, Kenneth R. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Via agrobacterium
C435S006120, C435S091200, C435S440000, C536S023100, C536S024300, C536S024330, C536S006000
Reexamination Certificate
active
06686515
ABSTRACT:
BACKGROUND OF THE INVENTION
The production of genetically altered plant species is of major agricultural and economic importance. In recent years, methods based on recombinant DNA techniques have led to the introduction of exogenous DNA from a variety of sources into the genomes of plant cells and explants. Regeneration of these genetically altered cells or explants into transgenic plants has dramatically increased the potential for discrete modifications of commercially relevant plant phenotypes.
A number of techniques exist for introducing exogenous DNA into plant cells, such as protoplasts, which are capable of subsequent regeneration, such as, microinjection of naked DNA, electroporation, Ca/PEG precipitation, and particle bombardment-mediated delivery, so called “biolistics.” Alternatively, it is possible to take advantage of the natural DNA transfer system of Agrobacterium to transfer exogenous DNA to plant chromosomes.
Agrobacterium mediated transformation relies on the ability of
A. tumefaciens
or
A. rhizogenes
to transfer DNA molecules called T-DNA to a host plant cell.
A. tumefaciens
and
A. rhizogenes
are the causative agents of the plant neoplastic diseases crown gall and hairy root disease, respectively. Agrobacteria, which reside normally in the soil, detect soluble molecules secreted by wounded plant tissues through a specialized signal detection/transformtion system. In the presence of these chemical signals, agrobacteria attach to the cell walls of wound exposed plant tissues. The agrobacteria then excise and transfer a portion of specialized DNA, designated T-DNA and delimited by T-DNA borders, to the host plant cell nucleus where it is integrated into the chromosomal DNA.
This DNA transfer system can be manipulated to transfer exogenous DNA situated between T-DNA borders to a host plant cell of choice. While Agrobacterium are typically restricted to infecting dicotyledonous species under natural conditions, by manipulating the conditions of infection, efficient transformation of monocots, including some crop species has been possible.
Common to the methods specified above is the integration of the exogenous DNA to a random site in the plant chromosome. While useful for many applications, random integration of transgenes leaves a number of difficulties. For example, the targeted disruption of an endogenous gene requires that integration occur at a specified locus in the host plant genome. Similarly, the ability to delete an endogenous gene and replace it with one that has been improved or modified, is of great commercial interest. In addition, great variability in expression levels exists between random integration events. The capacity to target insertion to a specific promoter or chromatin region conferring a desirable level or pattern of expression is a significant benefit that is gained by inserting a transgene at a predetermined site in the recipient genome.
Techniques available for directing transgenes to predetermined sites in the genomes of multicellular eukaryotes rely, on one hand, on homologous recombination between a transgene and an insertion site with which the transgene shares regions of sequence similarity; and on the other hand, on site specific recombinases. In the first case, large regions of sequence similarity flank a DNA sequence which introduces an alteration, most frequently a disruption, into a gene of interest. In the case of site specific recombinases, most commonly the Cre recombinase of bacteriophage P1 or the
Saccharomyces cerevisiae
FLP recombinase, DNA sequences lying between short repeated recognition sequences are inverted or exchanged. Again, while offering significant benefits, these methods have significant drawbacks. Homology mediated events generally require large (multi kilobase) regions of sequence similarity, while Cre or FLP recombinase mediated events are generally applicable only to sequences lying between the appropriate recognition repeats.
In prokaryotes, and in yeast, homologous recombination is a high efficiency event, and is the most common means of integrating an exogenous sequence into a bacterial or yeast chromosome. These homologous recombination events play a critical role in the repair of damaged DNA and rely significantly on the
E. coli
RecA protein and its homologues. RecA protein is a DNA binding protein that binds single stranded DNA with high efficiency regardless of nucleotide sequence. After binding of a single stranded DNA molecule and alignment with regions of similarity in a target sequence, RecA mediates strand exchange between two DNA substrates resulting in a homologous recombination event. The regions of similarity required in the RecA mediated event are 1-2 orders of magnitude smaller than those required by the multicellular eukaryotic processes described above.
The present invention provides solutions to many of the problems noted above, including providing site-specific integration of nucleic acids into plants. These and other advantages will be clarified by complete review of the following disclosure.
SUMMARY OF THE INVENTION
The present invention takes advantage of the recombinatorial properties of RecA and other recombinases to mediate the high efficiency integration of transgenes into predetermined sites within a host genome. Evolved recombinases with enhanced recombinatorial or other properties are used to mediate homologous recombination between exogenous DNA substrates and a desired site of insertion in a host chromosome. By allowing for integration into a desired insertion site without rigorous sequence requirements, the present invention significantly broadens the cases in which homologous recombination applies to the production of transgenic organisms. This facilitates the development of transgenic plants and animals with genetic alterations such as gene “knock-outs”, gene replacements, co-segregating transgene arrays, and novel exogenous/endogenous promoter-structural gene combinations in addition to randomly inserted transgenes.
In one aspect, the invention provides methods of evolving recombinase proteins which complement the Agrobacterium virE2 gene. To evolve recombinase proteins that complement virE2, one or more recombinase encoding nucleic acids are first diversified by any of a variety of methods. For example, such methods can entail supplying fragments of recombinase gene homologues derived from a variety of sources, recombining them in silico, in vitro or in vivo, and reconstructing a recombinant recombinase gene (by PCR based recursive elongation or other reconstruction methods), to generate a library of recombinase gene homologues. Recombination can be performed recursively for one or more cycle. The resulting library of recombinant nucleic acids is then screened to identify novel recombinase gene homologues that encode proteins which can substitute functionally for the Agrobacterium virE2 gene. In some embodiments, homologues of bacterial recA genes are given. In others, eukaryotic recombinases, such as Rad51 and Dmc1 are provided. Other embodiments provide for the use of evolved Agrobacterium Virulence proteins which have recombinase activity. A preferred embodiment provides for VirE2 proteins which have recombinase activity. In one embodiment, screening of the recombinase library is performed by expressing the recombinase protein homologues in a VirE2 deficient agrobacterium. In another embodiment, screening is performed by expressing the library in plant cells which are infected by VirE2 deficient agrobacterium.
The invention further provides methods of evolving RecA/VirE2 fusion proteins. These methods involve diversifying, e.g., recombining, recA and virE2 gene homologues in silico, in vitro or in vivo to establish a library of hybrid DNA molecules which encode RecA/VirE2 fusion proteins. This library is screened to identify RecA/VirE2 fusion proteins which maintain both RecA and VirE2 functional activities.
The present also invention provides for libraries of recombinant recombinase gene homologues and hybrid recA/virE2 genes.
In another aspect,
delCardayre Steven
Lassner Michael
Holman Christopher
Horlick Kenneth R.
Kruse Norman J.
Maxygen Inc.
Petithory Joanne R.
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