Method of Modifying Target Region in Host DNA and Selectable Marker Cassette

Abstract

A method of modifying a target region in a host DNA using a donor DNA: wherein the donor DNA having regions homologous to a 5′-side region outside of the target region in the host DNA, a 3′-side region outside of the target region in the host DNA and a first homologous recombination region inside of the target region in the host DNA, respectively, in this order, and further having a first selectable marker gene, an expression-inducing promoter and a second selectable marker gene expressed under the control of the expression-inducing promoter between the region homologous to the 3′-side region and the region homologous to the first homologous recombination region; which method has the steps of: a first step of performing homologous recombination between the donor DNA and the host DNA at the regions of the 5′-side region and the first homologous recombination region, to conduct selection of a host integrated with the donor DNA based on expression of the first selectable marker gene; and a second step of performing homologous recombination, within the host DNA integrated with the donor DNA by the first step, between two regions of the 3′-side region derived from the host DNA and the 3′-side region derived from the donor DNA, to conduct selection of a host whose target region is modified based on expression of the second selectable marker gene under an expression-inducing condition for the expression-inducing promoter; and a selectable marker cassette for use in the method.

Description

TECHNICAL FIELD [0001] The present invention relates to a method of modifying a target region in a host DNA and relates to a selectable marker cassette. BACKGROUND ART [0002] Drug-resistance genes have been used as a means for artificial selection of transformants in the genetic engineering field. Specifically, a desired transformant can be selected based on a drug-resistance gene whose expression confers drug resistance. Not only the drug-resistance genes but also, for example, auxotrophic genes are used as means for selecting transformants. These genes usable for selecting transformants are generally called selectable markers (selectable marker genes). [0003] Selectable marker genes are useful for selecting transformants. However, such a selectable marker gene, depending on its kind, unfavorably exhibits adverse effects on the environment, such as biological pollution of a wild-type microorganism by the selectable marker gene, if the selectable marker gene remains in a host cell. ...

AI Technical Summary

Benefits of technology

[0013] The present invention is to provide a method of modifying a target region in a host DNA; in which the method enables to modify the target region in the host DNA with a simple procedure without limiting the kind of a selectable marker (selectable marker gene) that can be used, and to modify the target region in the host DNA without leaving the selectable marker and the like used in modification steps in the modified host DNA; and to provide a selectable marker cassette for used therein.

[0038] According to the methods of the present invention, it is possible to modify the desired region in the host DNA with a simple procedure.

[0039] According to the present invention, neither the first selectable marker gene nor second selectable marker gene used in the modification process (including the deleting process and the replacing process) remain in the host DNA after modification. Accordingly, it is possible to prepare a desired transformant without limiting the range of the selectable marker genes that can be used. In particular when a plurality of regions in the host DNA are modified, the modifying process according to the present invention can be much simplified, compared with that by the conventional gene modification method that the selectable marker gene remains in the host, because any selectable marker gene can be used without restriction. In addition to the selectable marker gene, according to the present invention, the other regions (such as first homologous recombination region, promoter sequence, and the like) used in the modification process do not remain in the host DNA after modification. Thus, when a plurality of modification are repeatedly performed in the same host, the modification process according to the present invention can also be simplified.

[0040] Also, according to the present invention, it is possible to prevent the adverse effects on environment caused by residual of the selectable marker gene in the host. For example, if a drug-resistance gene is carried in a pathogenic strain (i.e., drug-resistant microbe), there is a concern about contamination of environment. The strain introduced with the drug-resistance gene is usually treated as a recombinant organism, and it demands to prevent the strain from spreading into the environment. Thus, the operation steps using such a strain more complicated, such as restriction in handling in production, use and storage of the strain, and demand to prevent diffusion of the strain. The present invention is effectively used for such the environmental containment caused by residual of the selectable marker gene.

Problems solved by technology

As described above, the conventional genetic manipulation method for modifying a host DNA and obtaining a transformant has a problem that a selectable marker is left in the transformant.

Thus, the method is not entirely satisfactory for improvement of operational efficiency.

In the conventional genetic manipulation method using a plasmid, it is difficult to introduce a large-size DNA segment or a lethal gene into a desired region of a host DNA.

Further, in the conventional method using a phage or cosmid, it is impossible to insert a plurality of genes into plural loci of a single cell.

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