The present invention relates to a method for selecting genetically transformed cells into which a desired nucleotide sequence has been incorporated by providing the transformed cells with a selective advantage without damaging the non-transformed cells, as well as to novel compounds for use in the method, and transformed cells.
It is well known that when new genetic material is to be introduced into a population of cells by transformation, only a certain number of the cells are successfully transformed. It is then necessary to identify the genetically transformed cells so that these cells can be separated from the non-transformed cells of the population. Identification and separation of the transformed cells has traditionally been accomplished using "negative selection", whereby the transformed cells are able to survive and grow, while the non-transformed cells are subjected to growth inhibition or perhaps even killed by a substance which the transformed cells, by virtue of their transformation, are able to tolerate.
For example, when a population of plant cells is transformed, selection of the transformed cells typically takes place using a selection gene which codes for antibiotic or herbicide resistance. The selection gene--which in itself generally has no useful function in the transformed plant (and may in fact be undesirable in the plant) is coupled to or co-introduced with the desired gene to be incorporated into the plant, so that both genes are incorporated into the population of cells, or rather into certain of the cells in the population, since it is difficult, if not impossible, in practice to transform all of the cells. The cells are then cultivated on or in a medium containing the antibiotic or herbicide to which the genetically transformed cells are resistant by virtue of the selection gene, thereby allowing the transformed cells to be identified, since the non-transformed cells--which do not contain the antibiotic or herbicide resistance gene in question--are subjected to growth inhibition or are killed.
These negative selection methods have, however, certain disadvantages. First of all, the non-transformed cells may die because of the presence of antibiotics or herbicides in the growth medium. As a result, when the population of cells is a coherent tissue there is a risk that not only the non-transformed cells but also the transformed cells may die, due to the fact that the death of the non-transformed cells may cut off the supply of nutrients to the transformed cells or because the damaged or dying non-transformed cells may excrete toxic compounds.
Another disadvantage of negative selection is that the presence of an unnecessary gene, for example antibiotic resistance, may be undesirable. There is concern among environmental groups and governmental authorities about whether it is safe to incorporate genes coding for antibiotic resistance into plants and microorganisms. This concern is of particular significance for food plants and for microorganisms which are not designed to be used in a closed environment (e.g. microorganisms for use in agriculture), as well as for microorganisms which are designed for use in a closed environment, but which may accidentally be released therefrom. While these concerns may prove to be unfounded, such concerns may nevertheless lead to government restriction on the use of antibiotic resistance genes in for example plants and it is therefore desirable to develop new methods for selecting genetically transformed cells which are not dependent on such genes.
A further disadvantage of negative selection is that plant tissues or cells treated with toxic substances become more susceptible to bacterial infection. This is a problem when Agrobacterium is used as a transformation vector, because the treated tissues or cells sometimes become overgrown with the bacteria even though antibiotics are used to prevent bacterial growth.
In addition, selection of cells or tissues using negative selection requires precise timing of expression of the introduced genes in relation to the selection process. If the transgenic cells are treated with a toxic compound before the detoxifying gene is expressed or before enough gene products are produced to ameliorate the action of the toxic compound, both the transgenic and the non-transgenic cells will be killed. If selection is performed too late, the selection of transgenic cells or tissues may be hindered by, for example, shoot or callus formation from non-transgenic cells or tissues which forms a barrier to the penetration of the compound used to select the transformed cells.
The above disadvantages are substantially overcome by the method according to the present invention, termed "positive selection" which for the first time makes it possible to identify and select genetically transformed cells without damaging or killing the non-transformed cells in the population and without co-introduction of antibiotic or herbicide resistance genes. In addition to the fact that the need for antibiotic or herbicide resistance genes is eliminated, the positive selection method according to the present invention is often far more efficient than traditional negative selection, and a combination of positive and negative selection gives a selection frequency of transgenic shoots as good as if not higher than that obtained using negative selection alone. Furthermore, the use of positive selection provides the advantage that a single gene may be used as both a reporter gene and a selection gene, resulting in simplification of vector constructions, more stable constructions and a 100% correlation between the expression of reporter and selection genes.
Positive selection may also eliminate the above-mentioned problems with regard to timing, since selective compounds may be produced as a consequence of the action of gene products, resulting from expression of the introduced gene, on particular substrates. Thus, the selective compound may accumulate as a consequence of expression of the selection gene, the selection effect appearing when a sufficient amount of the selective compound has been produced.
USPTO Backgrounds
USPTO Backgrounds