Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-05-17
2004-11-30
Forman, BJ (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S320100, C435S410000, C435S440000, C435S449000, C435S471000, C536S023100, C536S023400, C536S024300
Reexamination Certificate
active
06824983
ABSTRACT:
The present invention relates to a method for the mutagenesis of eukaryotic nucleotide sequences, preferably from plants, algae and/or fungi, and to a method of generating such genetically modified eukaryotic cells.
A possible method of elucidating gene functions is the inactivation of genes and the subsequent observation of the effects on the metabolism or the phenotype of the modified system.
In particular gene inactivation by insertion mutagenesis has proved difficult in eukaryotic systems. The success of gene inactivation by homologous recombination in the host genome depends essentially on the ratio in which undesired, site-independent recombination occurs over site-specific homologous recombination. This ratio differs widely in eukaryotic organisms. A sufficiently high efficiency of the method for generating a large number of mutants is only described for lower eukaryotic organisms and starting from genomic DNA. Efficacies of above 10% have previously only been demonstrated in yeasts (Grimm & Kohli, 1988, MGG 215, 87-93; Struhl 1983, Nature 305, 391-397), various filamentous fungi (Fotheringham & Holloman, 1989, Mol. Cell Biol. 9, 4052-4055; Kronstad et al., 1989, Gene 79, 97-106; Paietta & Marzluff, 1985, Mol. Cell Biol. 5, 1554-1559; Timberlake & Marshall, 1989, Science 244, 1313-1317) and in Dictyostelium discoideum (De Lozanne & Spudich, 1987, Science 236, 1086-1091).
Homologous recombination in plants has previously been reported from Arabidopsis thaliana (Kempin et al., Nature 1998, 389, 802-803) for the AGL5 gene, an MADS box factor and for the TGA3 locus (Miao & Lam 1995, Plant Journal 7, 359-365). Here, however, only individual events were observed, and these permit no information on the statistic frequency (Puchta 1998, Trends Plant Sci. 3 (3), 77-80). Phenotypic changes were not observed in these cases. In the case of the AGL5 gene, one of 750 analyzed plants had a site-specific mutation (which corresponds to a recombination rate of 0.13%); in the case of the TGA3 locus, one event was found out of 2580 mutants (which corresponds to a recombination rate of 0.04%). It has been reported for Lotus japonica that no homologous recombination event was identified out of 18974 transformants (Thykjaer et al. 1997, Plant Mol. Biol. 35, 523-530).
Using genomic fragments of single-copy genes, knockout mutants were obtained in the moss Physcomitrella patens as a consequence of homologous recombination (Schaefer et al., 1997, Plant Journal 11 (6): 1195-1206). The homologous recombination rate was 90%. Again, no change in phenotype was observed in this case.
The utilization of genomic nucleotide sequences for generating mutants is disadvantageous inasfar as the DNA fragments may comprise a variety of operable units or linkages. Operable unit or linkage is understood as meaning the sequential arrangement of individual units and their linkage to each other, such as promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its intended function upon expression of the coding sequence. Site-unspecific insertion may have such a random modifying effect on, for example, promoters that a regulator with novel characteristics arises.
The analysis of the mutants may reveal further problems. If, for example, two genes are disrupted by a nucleic acid fragment, no definite conclusion regarding the involvement of a gene can subsequently be drawn since the contribution of an individual gene to the effect to be observed cannot be assessed. This would first have to be tested by more complicated analyses, such as, for example, sequencing of the genomic fragments used, and by determining the type of insertion in the plant and its effect on the genomic arrangement of modified gene segments.
The inactivation of genes by the insertion mutagenesis of genomic DNA by means of homologous recombination by known methods furthermore has a further considerable disadvantage. Thus, for example, the analysis of the entire genome of a host system requires the complicated construction of several thousand individual constructs of genomic origin, which constructs must also be retransformed individually into the host cell, before analysis of the mutants obtained by homologous recombination is made possible. Such genomic mutation approaches are thus not very economical and unsuitable for a routine throughput of experimental set-ups of substantial size, for example with the aim of obtaining a “saturated” mutagenesis of the entire host genome.
It is an object of the present invention to provide a simple and efficient method for the mutagenesis in eukaryotic cells which no longer has the abovementioned disadvantages.
We have found that this object is achieved by a method for the mutagenesis of eukaryotic nucleotide sequences in which a genomic nucleotide sequence and/or cDNA sequence from eukaryotic organisms is transferred into a microorganism, the eukaryotic nucleotide sequence is genetically modified in the microorganism by sequence-independent insertion mutagenesis, and this genetically modified eukaryotic nucleotide sequence is subsequently isolated from the microorganism. Only one transposition event takes place per transferred eukaryotic nucleotide sequence. The transfer of eukaryotic cDNA is preferred.
In a further variant of the method according to the invention, the sequence-independent insertion mutagenesis is achieved by cointegrate formation during the conjugation between two microorganisms. Here, the eukaryotic nucleotide sequence is transferred into a microorganism which then acts as donor cell; the eukaryotic nucleotide sequence is subsequently mutated in the microorganism by sequence-independent insertion mutagenesis, during which process a cointegrate is first formed, the cointegrate formed is then transferred into another microorganism (receptor) via conjugation, the cointegrate is broken down in the receptor, during which process the eukaryotic nucleotide sequence is genetically modified by insertion of a heterologous nucleotide sequence, and this genetically modified eukaryotic nucleotide sequence is isolated from the microorganism. Heterologous DNA is to be understood in accordance with the invention as meaning DNA which does not naturally occur in eukaryotic organisms, i.e. which originates from prokaryotic organisms or, if appropriate, from viruses or phages, and which is replicated in microorganisms. In a preferred variant of the method according to the invention, cDNA sequences from eukaryotic organisms, preferably from plants, algae and/or fungi, are employed.
It is essential for the present method that the mutagenesis in the microorganism takes place with a high degree of efficiency, preferably with a relative frequency post-selection in a range of from approximately 90 to 100%, especially preferably of more than 90 to 99%, in particular 99.9%. In accordance with the invention, only one insertion event takes place per nucleotide sequence.
Since, in accordance with the invention, the mutagenesis of the eukaryotic nucleotide sequence takes place in a sequence-independent manner, that is to say randomly, by insertion of a heterologous nucleotide sequence, the method according to the invention is advantageously distinguished by the fact that the eukaryotic nucleotide sequence can be mutated without it being necessary to know its exact nucleotide sequence or specific restriction cleavage sites. Based on a selected region of a eukaryotic nucleotide sequence, a multiplicity of different (chimeric) eukaryotic nucleotide sequences, which merely differ from each other by the insertion site of the heterologous nucleotide sequence in the eukaryotic DNA, are generated in accordance with the invention by the sequence-independent mutagenesis.
Furthermore, the method according to the invention is also suitable for the mutagenesis of a very large number of different eukaryotic nucleotide sequences as are present for example in the form of a eukaryotic genetic library. A considerable advantage of the method according
Duwenig Elke
Freund Annette
Guitton Marie-Christine
Rak Bodo
Reski Ralf
BASF Plant Science GmbH
Forman BJ
Morrison & Foerster / LLP
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