Trap vectors and gene trapping using the same

Abstract

A trap vector containing a loxP sequence composed of inverted repeat sequence 1, a spacer sequence and inverted repeat sequence 2 in this order, the loxP sequence being a mutant loxP wherein a part of the inverted repeat sequence 1 or 2 is mutated.

Description

[0001]This application is a Divisional of co-pending application Ser. No. 10 / 030,658 filed on Jan. 11, 2002 and for which priority is claimed under 35 U.S.C. § 120. Application Ser. No. 10 / 030,658 is the national phase of PCT International Application No. PCT / JP00 / 02916 filed on May 2, 2000 under 35 U.S.C. § 371. The entire contents of each of the above-identified applications are hereby incorporated by reference.TECHNICAL FIELD[0002]The present invention relates to random mutation ES clone technology using gene trapping.BACKGROUND ART[0003]It is said that structural analysis of human genome will be completed in or before 2003 as the human genome project is progressing well. Now, the age of isolating genes one by one and analyzing their structures separately seems to be over, and we have come into the age of “structural analysis” of genome.[0004]With the nucleotide sequence of genome alone, however, information on functions is insufficient. Thus, a novel analysis system for function...

AI Technical Summary

Benefits of technology

[0051]Generally, exon trap vectors are composed of a reporter gene with a splice acceptor alone, a drug selection marker gene and a plasmid. Only when these vectors are integrated downstream of a mouse endogenous gene, the reporter gene is expressed. This means that it is possible to know the vector's integration into an endogenous gene by monitoring the expression of the reporter gene in the trap vector. If a plasmid such as pUC19 has been linked to the trap vector, the trapped endogenous gene can be isolated by the technique called plasmid rescue. “Plasmid rescue” is a technique for recovering a gene of interest by selection with ampicillin, etc. of those cells transformed with electroporation or the like (FIG. 2E). Furthermore, since the endogenous gene is disrupted at the time of trapping, knockout mice can be produced immediately. Further, since the reporter gene is expressed under the control of the expression regulatory region of the endogenous gene, the tissue specificity and time specificity of the gene can be analyzed easily.

[0060]Actually, when a mutant loxP (hereinafter, sometimes referred to as “mutant lox”) such as lox71 has been integrated into ES cells in advance, and a plasmid containing other mutant loxP (e.g. lox66) is introduced thereinto, the plasmid is integrated into the genome. Therefore, if this lox71, for example, has been integrated into a gene trap vector in advance, it becomes possible to insert any desired gene later by using lox66. Thus, according to the present invention, it has become possible to replace the trapped gene with a gene into which a subtle mutation(s) has (have) been introduced or a human gene.

[0079]The vector shown in (b) above is designated “U8delta”. U8delta is obtainable by deleting the splice acceptor from U8. This vector has a structure in which lox71 is linked before the reporter β-geo and loxP after β-geo. This vector was given such a structure because the intermediate IRES and β-geo can be removed completely by transiently expressing Cre after the vector has been integrated. As a result, plasmid pUC is located close to the mouse endogenous gene which was located upstream of the plasmid. Thus, the mouse endogenous gene can be isolated easily.

[0080]The vector shown in (c) above is designated “pU-Hachi”. This vector is composed of SA-lox71-IRES-M-loxP-pA-PV-SP. pU-Hachi vector is derived from pGT1.8IRES β-geo, and contains SA sequence from mouse En-2 gene and β-geo sequence linked to encephalomyocarditis virus-derived IRES sequence. A BamHI fragment of lox71 is inserted into the BglII site of pGT1.8IRES β-geo. Then, a plasmid was constructed by inserting an SP sequence, a loxP sequence, and poly A addition signal from mouse phosphoglycerate kinase-1 (PGK) into a modified vector from which lacZ sequence has been removed. The SP sequence is used to protect the 3′ end of the trap vector. pU-Hachi is obtainable by inserting a SalI fragment of SA-IRES-lox71-β-geo into the SalI site of the above plasmid.

[0095]Chimeric animals are produced by standard methods (FIG. 9). The species of chimeric animals produced in the present invention is not particularly limited. For example, mouse, rat, guinea pig, rabbit, goat, sheep, pig, dog or the like may be enumerated. In the present invention, mouse is preferable because of its easy handling and propagation.

[0102]The animals that may be used in the present invention include mouse, rat, guinea pig, rabbit, goat, sheep, pig and dog. Preferably, mouse is used in the invention because of its easy handling and propagation.

Problems solved by technology

With the nucleotide sequence of genome alone, however, information on functions is insufficient.

Further, although one of the major goals of human genome analysis is to elucidate causative genes in human diseases, such diseases cannot be explained with the structures of causative genes alone.

The analysis of the functions of those elements to which the transcription factor binds is extremely difficult at present because a number of those elements exist in the regulatory region of one gene.

First, this method requires too much time. In the production of knockout mice, it is the rate-determining step to isolate knockout ES clones generated through homologous recombination using ES cells. Even a skillful researcher needs at least three months for isolating a knockout ES clone. Thus, only four genes can be knocked out in one year. Accordingly, in the case of introducing each one mutation into 105 genes, 2,500 researchers are required for one year. It is estimated that approximately 1,000 lines of knockout mice are produced in one year in the world. This means that it would take 100 years to produce 105 knockout ES clones. This is so unrealistic compared to the advance in the structural analysis of human genome that is to be completed in 2003.

Secondly, this method requires too much cost. At least 2 to 4 million yen is necessary to produce one line of knockout mouse excluding personnel expenses and depreciation expenses. Thus, production of 105 simple knockout mice requires 200 to 400 billion yen.

As described above, the conventional homologous recombination using ES cells has problems, and genome is vast.

However, the number of genes in genome is limited.

However, each of the methods using a mutagen and the method using gene trapping has an advantage(s) and a drawback(s) (Table 1).

According to the ENU method, production of mutant mice is easy, but establishment of individual mutant lines is not easy because segregation by crossing should be conducted.

Thus, the ENU method requires complicated operations.

Besides, generally, methods using a mutagen such as chlorambucil need large breeding rooms.

Thus, such methods require much expenses and labor.

However, ordinary researchers can hardly use this service because of the following reasons.

Briefly, it is not sure whether an endogenous gene is disrupted or not even if the gene is trapped; it is not clear whether germline chimeric mice can be produced; an additional charge is required for the production of chimeric mice; and considerable charges are required for using the service.

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