Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-12-23
2002-09-03
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S288000, C800S317200, C800S320100, C800S278000, C435S418000, C435S419000, C435S320100, C435S468000, C536S023200, C536S023700
Reexamination Certificate
active
06444878
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a selection method.
The present invention also relates to an enzyme and a nucleotide sequence coding for same that are useful in a selection method.
In particular, the present invention relates to a method for the selection (e.g. identification and/or separation) of genetically transformed cells and compounds and genetic material for use in the method.
BACKGROUND OF THE INVENTION
It is well known that when a nucleotide sequence of interest (“NOI”) is to be introduced into a population of cells by transformation, only a certain number of the cells are successfully transformed, i.e. receive the NOI. It is then necessary to identify the genetically transformed cells so that these cells may be separated from the non-transformed cells in the population.
A common technique for a selection method includes introducing transformed cells and non-transformed cells into a medium that comprises a substance which the transformed cells are able to tolerate. In that medium the transformed cells are able to survive and grow, while the non-transformed cells are prone to growth inhibition and, in some cases, are killed.
To date, if a population of plant cells has been subjected to genetic transformation, selection of the transformed cells typically takes place using a selection gene which codes for antibiotic resistance or herbicide resistance. The selection gene is coupled to or co-introduced with the NOI to be incorporated into the plant in question, so that both of the two genes are incorporated into some or all of the population of cells.
As not all of the cells may have been transformed, the cells are then cultivated on or in a medium containing the respective antibiotic or herbicide to which the genetically transformed cells are resistant by virtue of the selection gene. In this medium, the transformed cells are able to grow and thus be identified out of the total cell population, since the non-transformed cells—which do not contain the antibiotic or herbicide resistance gene in question—have an inhibited growth or even are killed.
These selection methods which rely on the use of antibiotics or herbicides suffer from a number of disadvantages. For example, there is concern amongst some people, such as environmental groups and governmental authorities, as to whether it is environmentally safe to incorporate genes coding for antibiotic resistance and/or herbicide resistance into plants and micro-organisms. This concern is of particular significance for food plants and for micro-organisms which are not designed and/or intended to be used in a closed environment (e.g. micro-organisms for use in agriculture), and also for micro-organisms which are designed for use in a closed environment but which may be released from the closed environment. While these concerns may prove to be unfounded, each concern may nevertheless lead to governmental restrictions on the use of antibiotic resistance genes and/or herbicide resistance genes in e.g. plants.
It is therefore desirable to develop new methods for selecting genetically transformed cells or organisms (or parts thereof) comprising such.
SUMMARY OF THE INVENTION
According to a first aspect of the present invention there is provided a selection method for selecting from a population of cells one or more selectable genetically transformed cells, wherein the population of cells comprises selectable genetically transformed cells and possible non-transformed cells; wherein each of the selectable genetically transformed cells comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for both the selectable genetically transformed cells and the non-transformed cells; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to the non-transformed cells; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cells; the method comprising the step of introducing the population of cells to a medium, wherein the medium optionally comprises a high concentration of the component or the metabolic derivative thereof, and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cells; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a second aspect of the present invention there is provided a composition comprising a population of cells comprising selectable genetically transformed cells and possible non-transformed cells; and a medium, wherein each of the selectable genetically transformed cells comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for both the selectable genetically transformed cells and the non-transformed cells; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to the non-transformed cells; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cells; the medium optionally comprising a high concentration of the component or the metabolic derivative thereof, and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cells; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a third aspect of the present invention there is provided a population of cells comprising selectable genetically transformed cells and possible non-transformed cells; wherein each of the selectable genetically transformed cells comprises a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for both the selectable genetically transformed cells and the non-transformed cells; wherein the component or the metabolic derivative thereof when present in a high concentration in a medium is toxic to the non-transformed cells; wherein the first nucleotide sequence codes for a gene product capable of converting the component or the metabolic derivative thereof when present in a high concentration in a medium to a nutrient for the selectable genetically transformed cells; and either wherein the component or the metabolic derivative thereof is a source of both carbohydrate and nitrogen for the selectable genetically transformed cells; or wherein if a portion of the component serves as a metabolic substrate and is metabolically converted to a derivatised substrate, then that derivatised substrate is capable of providing an allosteric effect on the gene product.
According to a fourth aspect of the present invention there is provided a selectable genetically transformed cell comprising a first expressable nucleotide sequence and optionally a second expressable nucleotide sequence; wherein a component or a metabolic derivative thereof when present in a low concentration in a medium is a nutrient for the selectable genetically transformed cell and a non-transformed cell; wherein the component or the
Bojsen Kirsten
Donaldson Iain A.
Jorgensen Kirsten
Jorsboe Morten
Danisco A/S
Fox David T.
Knobbe Martens Olson & Bear LLP
Kruse David H
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