Method for gene amplification

The present invention relates to a method for amplifying gene at high speed and a method for producing proteins by using the amplified gene.

Gene amplification with cultured animal cells (Reference 1 and the like) accompanies several complications such as (1) time consuming (a half to one year), (2) presence of clones without amplification, and (3) empirical procedures with unexplained mechanism. On the other hand, there is no system of gene amplification with yeast. Although plasmids are generally used for the purpose, increase in copy number beyond a certain threshold is difficult.

The system of the present invention is based on the replication referred to as DRCR (Double Rolling-Circle Replication) induced by biological potency called as BIR (Break-Induced-Replication) (Reference 2-4). It is conceivable that a chromosome breakage is rescued itself by the following steps; i.e. the broken chromosome finds homologous sequence, invades into it, forms a replication fork, and consequently starts DNA replication. All living organisms might involve such ability.

Moreover, it is reported that natural circular DNA accompanies DRCR by recombination (Reference 5).

Reference 1: Japanese Patent Gazette 8-504585 (WO94/14968)

Reference 2: WO2005/061703

Reference 3: PNAS, vol. 98, no. 15, 8255-8262 (Jul. 17, 2001)

Reference 4: Genes Dev 12, 3831-3842 (1998)

Reference 5: Cell. 1986 Aug. 15; 46 (4): 541-550

The present invention provides a double-stranded DNA constructed specially for high speed gene amplification, a method for gene amplification thereby and protein production thereby. The present invention is characteristic in full artificially designed system of gene amplification, the potential of higher amplification efficiency by synchronous culture, short period for amplification (probably one generation) and well elucidated mechanism of amplification.

The amplification system of the present invention utilizes a type of DNA replication referred to as double rolling-circle replication (DRCR). The type of replication is able to amplify DNA explosively in a single cell cycle. It is assumed that the amplified products are maintained intracellularly after termination of DRCR by recombination and the like. The present inventors utilized a site-specific recombinase such as Cre-lox system and its target sequence in order to induce DRCR efficiently. More specifically, the present inventors constructed a replication unit (ex. FIG. 3) in yeast and were able to succeed in inducing DRCR by utilizing a recombination generated by a site-specific Cre recombinase (hereinafter, referred to as “Cre”) during progress of a replication fork between a pair of lox sequences and to accomplish the present invention.

Namely, the present invention is a double-stranded DNA represented by a-b-c-d or a-c-b-d, wherein one of a and b is a double-stranded DNA fragment comprising a first target sequence of a site-specific recombinase, and the other is a double-stranded DNA fragment comprising an inverted sequence of said first target sequence; and one of c and d is a double-stranded DNA fragment comprising a second target sequence of the site-specific recombinase and the other is a double-stranded DNA fragment comprising an inverted sequence of said second target sequence; a replication origin and at least one target gene to be amplified are inserted anywhere between a and d; and arbitrary DNA sequences may be inserted among above fragments.

Additionally, the present invention is a recombinant vector comprising the double-stranded DNA, and is also a transformant, which is introduced with the double-stranded DNA.

Moreover, the present invention is a set of double-stranded DNA comprising a double-stranded DNA fragment represented by e-a-A-b-f and a double-stranded DNA fragment represented by g-c-B-d-h, wherein one of a and b is a double-stranded DNA fragment comprising a first target sequence of a site-specific recombinase, and the other is a double-stranded DNA fragment comprising an inverted sequence of said first target sequence; and one of c and d is a double-stranded DNA fragment comprising a second target sequence of the site-specific recombinase and the other is a double-stranded DNA fragment comprising an inverted sequence of said second target sequence; each of letters from e to h is a double-stranded DNA fragment of at least 50 bp in size, which are arranged on a chromosome or an extrachromosomal element that is a host for integration of the set of double-stranded DNA in order of e, f, a replication origin of the chromosome element or the extrachromosomal element, g and h; at least one of A and B represents the target gene to be amplified; and said replication origin or a part of it may be included in f or g; and an arbitrary DNA sequence may be inserted among these.

The present invention is also a set of recombinant vectors, wherein each vector contains each of two kinds of the double-stranded DNA, and is also a transformant or transfectant, which is introduced with two kinds of the double-stranded DNA, wherein said replication origin locates on a host chromosome or an extrachromosome.

The present invention is also a method for amplifying the target gene, comprising the steps of preparing the transformant or the transfectant and affecting said transformants with the site-specific recombinase; and is a method for manufacturing a protein encoded by the target gene, comprising a step of culturing transformed or transfected cells obtained by the above method.

The amplification system of the present invention has an excellent property in establishing efficient system for producing proteins. DRCR is capable of amplifying a target gene rapidly during a single cell cycle. Since the amplification mechanism is well elucidated, reliable amplification of a target gene is prospective. Although the present example was constructed in yeast not animal cells, it is possible to produce highly amplified products at 10 to 100 times higher frequency than a conventional system of animal cultured cells. Furthermore, the present system can be applied to primary cultured cells, in which gene amplification by drug selection has not been observed. Therefore, it is possible to apply gene amplification to targeting cells of gene therapy, and to enhance and sustain the expression of introduced gene.