Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-07-07
2002-10-15
Ketter, James (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S463000, C435S455000, C536S023100, C424S093100
Reexamination Certificate
active
06465254
ABSTRACT:
TECHNICAL FIELD
This invention relates to a mutant loxP site and applications thereof. More particularly, the present invention relates to the mutant loxP site, in which a specific recombination between the mutant loxP site and a wild-type loxP site can not occur, but a specific recombination between mutant loxP sites can occur in each other, and gene replacement using the said mutant loxP site.
BACKGROUND ART
It is not so easy to integrate any gene into specific sites of animal virus or chromosome of animal cells of the higher eucaryotes or to delete specific gene therefrom. A conventional method for gene integration into the specific site of chromosome of animal cells is, for example, that cells are transformed with plasmid DNA, to which DNA having the same site with the site of chromosome to be intended to integrate is ligated with both sites of the objective gene, to obtain the cells, to which the objective gene is integrated by homologous recombination. Frequency of homologous recombination is, however, extremely low. To that end, the objective gene and drug resistant gene should be simultaneously integrated and selected by drug. Consequently, several months have to be required to obtain the objective cells. Further, although preparation of recombinant animal virus, to which the objective gene is integrated, is slightly easier than the previously described case of chromosome of cells, however even in case that, for example, the recombinant adneovirus is constructed, various treatments including homologous recombination by using plasmids, to which objective gene is integrated, as well as cloning, selection and growth of the recombinant virus are required (Bett et al., Proc. Natl. Acad. Sci., 91: 8802-8806, 1999 and Miyake et al., ibid. 93: 1320-1324, 1996).
One of reasons why the gene manipulation of specific sites in chromosome of animal cells and the construction of recombinant virus are difficult is using homologous recombination with low frequency. Contrary to that, if it can be used enzymes, which can specifically recognize DNA Sequence, as like restriction enzymes used for construction of plasmid or bacteriophage, it is expected to improve the efficiency of the gene manipulation on cell chromosome. Example of such the enzyme is recombinase Cre derived from bacteriophage P1 of
E. coli.
Cre is a specific DNA recombinase, which recognizes specific nucleotide sequence (loxP site) and conducts total processes including DNA strand cleavage, strand exchange and ligation of each DNA strand within this site (Sternberg et al., J. Mol. Biol., 150: 467-468, 1981; Abremski et al., J. Biol. Chem., 259: 1509-1514, 1984; and Hoess et al., Proc. Natl. Acad. Sci., 81: 1026-1029, 1984). In case that two loxP sites of the same direction exist within the same DNA molecule, DNA sequence between them is excised to form circular molecule (DNA excision reaction), or on the contrary in case that two loxP sites exist in the different DNA molecules, and the one is a circular DNA, the circular DNA is inserted into the other DNA molecule through loxP site (insertion reaction). Although Cre and loxP site were found in bacteriophage, the specific DNA recombination reaction is known to function not only in the procaryotes but also in the eucaryotes including animal cells and in the animal viruses. Examples of excision reactions are cultured animal cells (Sauer et al., Nucleic Acids Res., 17: 147-161, 1989 and Kanegae et al., Gene, 181: 207-212, 1996), animal viruses (Sauer et al., Proc. Natl. Acad. Sci., 85: 5166-5170, 1988; Anton et al., J. Virol., 69: 4600-4606, 1995; and Kanegae et al., Nucleic Acids Res., 23: 3816-3821, 1995), and transgenic mice (Lakso et al., Proc. Natl. Acad. Sci., 89: 6232-6236, 1992; Orban et al., ibid., 89: 6861-6865, 1992; Gu et al., Cell, 73: 1155-1164, 1993 and Gu et al., Science, 265: 103-106, 1994).
In addition, if the insertion reaction is applied, any gene can be inserted into the chromosome of animal cells or viral genome, in which loxP site exists previously, but the frequency of insertion is extremely low (Fukushige et al., Proc. Natl. Acad. Sci., 89: 7905-79029, 1992 and Sauer et al., Proc. Natl. Acad. Sci., 84: 9108-9112, 1987), consequently it is not practicable. Because, the insertion and excision are irreversible reactions, consequently if two loxP sites are existed in the identical DNA molecule as a result of insertion reaction, the excision reaction immediately occurs, moreover a reaction equilibrium lies overwhelmingly so far to the excision reaction.
In order to increase frequency of the insertion reaction, trials on using loxP site (mutant type), which is different from the original nucleotide sequence of loxP site (wild-type), were performed. The loxP site consists of DNA sequence of 34 bp (SEQ ID NO: 1), as shown below in both the 5′ to 3′ and the 3′ to 5′ direction. Among them, 8 bp sequence between two 13 bp inverted repeats is designated as spacer region, and recombination of DNA strand is known to be carried out within the spacer region (Hoess et al., J. Mol. Biol., 181: 351-362, 1985).
loxP site (34bp)
<------------------------>
12345678
5′-ATAACTTCGTATA ATGTATGC TATACGAAGTTAT-3′
3′-
TATTGAAGCATAT
TACATACG
ATATGCTTCAATA
-5′
Inverted Spacer Inverted
Repeat Region Repeat
(13bp) (8bp) (13bp)
It was shown that the specific DNA recombination reaction between loxP site (mutant loxP site), in which a base at position 7 in the spacer region is substituted from G (guanine) to A (adenine), and wild-type loxP sequence can not occur, but the specific DNA recombination reaction between two mutant loxP sites can occur (Hoess et al., Nucleic Acids Res., 14: 2287-2300, 1986).
Trials that a gene located between mutant loxP site and wild-type loxP site in the DNA molecule is inserted between mutant loxP site and wild-type loxP site in the other DNA molecule or replaced by the other gene between them, are carried out. Examples of these trials are replacement of a gene on the plasmid vector by a gene on the bacteriophage gene (Waterhouse et al., Nucleic Acids Res., 21: 2265-2266, 1993), insertion of a gene on the phagemid vector to the plasmid vector (Tsurushita et al., Gene, 172: 59-63, 1996) and replacement of a gene on the plasmid vector by a gene on the chromosome of animal cells (Bethke et al., Nucleic Acids Res., 25: 2828-2834, 1997).
These trials were, however, performed by using only one mutant loxP site, i.e. the loxP site in which a base at position 7 of the spacer region was substituted from G (guanine) to A (adenine) (mutant loxP site), and the fact that whether it is preferable or not, is unknown, because the recombination reaction between the mutant loxP site in the said sequence and the wild-type loxP site does not occur. Further, in the above all three trials, the experimental systems, in which the drug resistant gene itself or the drug resistant gene together with objective gene is inserted and as a result, the recombinants having DNA molecules accompanied with the objective recombination can only acquire drug resistance and amplyfy, are used. Consequently, even if efficiency of the actual gene insertion (gene replacement) is low due to the recombiantion between the loxP site with incomplete mutation and the wild-type loxP site, such the experimental result is biased by the selection with drug resistance, and the apparent reaction efficiency may possibly be expressed too high.
Actually, as a result of direct and quantitative measurement found by us, in the mutant loxP
Saito Izumu
Tanaka Keiji
Ketter James
Li Janice
Sughrue & Mion, PLLC
Sumitomo Pharmaceuticals Company Limited
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