Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-09-14
2004-05-11
Falk, Anne-Marie (Department: 1636)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S325000
Reexamination Certificate
active
06734295
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is based upon and claims the benefit of priority from the prior Japanese Patent Application No. 11-264364, filed Sep. 17, 1999, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
The site-specific recombination is a phenomenon found in the process in which &lgr; phage DNA is integrated into a host chromosome. The site specific recombination is mediated by a recombination enzyme called recombinase which catalyzes recombination by recognizing a relatively short specific sequence, whereas the homologous recombination is performed by pairing long homologous nucleic acids. In this respect, the site specific recombination is a biological event completely different from the homologous recombination.
The site-specific recombination can be used to selectively recombinize a gene construct having a desired gene bound thereto, thereby knocking-in or knocking-out the desired gene. Therefore, the site-specific recombination is a very useful technique, especially in the field of embryological engineering for knocking-out or knocking-in a specific gene in a time- or location-controlled manner.
Now, referring to
FIG. 1
, the mechanism of the site-specific recombination and its application will be explained in brief.
As shown in
FIG. 1
, unlike the homologous recombination which is initiated with DNA pairing, the site-specific recombination is triggered with binding of recombinase 1 to a specific sequence 3 in DNA 2 to form a DNA-protein complex 5. The recombinase 1 bound to DNA 2 recognizes and binds to a specific sequence 4 which is present in the same DNA 2 or a different DNA and which has the same nucleotide sequence as the specific sequence 3.
FIG. 1
shows the case where the specific sequence 3 and 4 are present in the same DNA. The recombinase bound to the specific sequence 3 and 4 catalyses a cleaving/rebinding reaction of single-strand DNA. More specifically, the reaction is performed by two steps: sequentially cleaving the 3′ ends of the specific sequence 3 and 4; and binding a cleaved portion of the specific sequence 3 to a site A′ and a cleaved portion of the specific sequence 4 to a site A.
As shown in
FIG. 1
, in the case where the specific sequences are present in the same DNA, the DNA is cleaved into two, one a straight DNA, and the other a cyclic DNA, by the site-specific recombination. The cyclic DNA falls out from the original DNA.
Therefore, if a gene construct having a desired gene arranged to be fallen off as the cyclic DNA, and a recombinase gene are introduced into a chromosome, and then, the recombinase gene is expressed in a time-controlled and/or location-controlled manner, only the corresponding gene is knocked-out in the time-controlled and/or location-controlled manner.
Alternatively, a gene construct and a recombinase can be introduced into a chromosome to selectively “knock-in” a desired gene in the gene construct. In the gene construct, the desired gene is placed downstream of a first specific sequence and a promoter is placed upstream of a second sequence such that the gene is transferred to be flanked with the promoter after a recombination process in which an intervening sequence between the promoter and the gene is fallen off. Accordingly, knock-in is achieved in time and location controlled manner by expression of the recombinase.
As the recombinase which catalyses the site-specific recombination, FRT recombinase and FLP recombinase which are derived from a yeast, and phage-derived Cre recombinase have been found. However, the yeast-derived FRT and FLP recombines do not work well in mammalian cells.
In contrast, the Cre-loxP system consisting of Cre recombinase and a loxP sequence, which is specifically recognized by Cre recombinase, can be applied to mammalian cells. Therefore, the Cre-loxP system is used to initiate the site-specific recombination in mammals.
However, since the Cre recombinase is a bacteriophage-derived protein, the codons in the Cre recombinase is not translated efficiently in mammalian cells. Therefore, the Cre recombinase has a drawback in that it is expressed insufficiently.
The present invention is made to overcome the aforementioned drawback associated with the phage-derived Cre recombinase gene. An object of the present invention is to provide a modified Cre recombinase gene for mammals that is expressed in mammalian cells, tissues, organs, or body several times as abundantly as the phage-derived Cre recombinase gene.
BRIEF SUMMARY OF THE INVENTION
To solve the aforementioned object, the present invention provides a modified Cre recombinase gene for mammal (SEQ ID NO:1).
The present invention is to provide a modified Cre recombinase gene for mammals having a nucleotide sequence represented by (SEQ ID NO:1).
The modified Cre recombinase gene for mammals of the present invention encodes the same Cre recombinase protein derived from a bacteriophage P1 having an amino acid sequence represented by (SEQ ID NO:2). However, all codons are modified into those most frequently used in swine DNA. Therefore, the modified Cre recombinase gene of the present invention is expressed more abundantly in mammals compared to the phage-derived Cre recombinase gene.
Furthermore, the present invention provides a method of knocking-in or knocking-out a desired gene by the modified Cre recombinase gene in a location-controlled and/or time-controlled manner.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.
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“Uniform Vascular-Endothelial-Cell-Specific Gene Expression in Both Embryonic and Adult Transgenic Mice”, T
Miyagawa Shuji
Okabe Masaru
Falk Anne-Marie
President of Osaka University
Qian Celine
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