JP2004298180A - Mouse with altered genome - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To understand physiological role and elucidation of pathophysiology of enzymes associated with sugar chain modification bonding to form an α-bonding on 1st position of fucose to 6th position of N-acetylglucosamine on the N-glycoside linkage complex type sugar chain reducing end, and to provide an animal model useful for development of a medicine which targets the enzymes associated with sugar chain modification bonding to form the α-bonding on the 1st position of fucose to the 6th position of N-acetylglucosamine on the N-glycoside linkage complex type sugar chain reducing end. <P>SOLUTION: This invention includes a mouse or its offspring having an enzyme with reduced or deleting activity wherein the enzyme is associated with sugar chain modification bonding to form the α-bonding on the 1st position of fucose to the 6th position of N-acetylglucosamine on the N-glycoside linkage complex type sugar chain reducing end. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a genomic strain such that the activity of an enzyme involved in sugar chain modification in which the position 1 of fucose is α-linked to the position 6 of N-acetylglucosamine at the reducing end of N-glycoside-linked complex type sugar chain is reduced or deleted. Is a mouse or progeny thereof.

As an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the reducing end of an N-glycoside-linked complex type sugar chain, in mammals, α1,
6-fucosyltransferase is known (Non-Patent Document 1). The structure of the α1,6-fucosyltransferase gene (Non-patent document 2) was clarified in 1996 (Non-patent document 3; Non-patent document 4; Patent document 1). Although the enzyme activity of α1,6-fucosyltransferase has been confirmed in many organs, it has been reported that it is relatively high in the brain and small intestine (Non-Patent Document 5; Non-Patent Document 6). It has been pointed out that fucose-modified sugar chains play an important role in retinal formation as a physiological function, and attention has been paid to the relationship between retinal formation and regulation of α1,6-fucosyltransferase expression ( Non-Patent Document 7). In blood coagulation, platelet-derived α1,6-
The role of fucosyltransferase has been pointed out (Non-Patent Document 8). Also,
It has also been reported that the modification of fucose to the sugar chain structure of immunoglobulin IgG1 changes the binding between IgG1 and FcγRIIIa, and changes the antibody-dependent cytotoxic activity of the antibody itself (Non-Patent Document 9, Non-Patent Document 9). 10). Regarding the relationship with the disease state, in some diseases such as liver cancer and cystic fibrosis, an increase in α1,6-fucosyltransferase activity and an increase in the ratio of the enzyme reaction product have been observed. Relevance has been pointed out (Non-Patent Document 11, Non-Patent Document 12). Transgenic mice overexpressing α1,6-fucosyltransferase have also been produced, and it has been reported that in the produced transgenic mice, steatosis-like degeneration is observed in the liver and kidney (Non-Patent Document 13).

However, a transgenic genome modified so that the enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked complex type sugar chain is reduced or deleted. Although a transgenic non-human animal is disclosed in Patent Document 2, there is no report that a mouse whose genome has been modified has actually been produced.
Biochem. Biophys. Res. Commun., 72, 909 (1976) EC 2.4.1,68 J. Biol. Chem., 271, 27817 (1996) J. Biochem., 121, 626, (1997) Int. J. Cancer, 72, 1117 (1997) Biochim. Biophys. Acta., 1473, 9 (1999) Glycobiology, 9, 1171 (1999) Biochem. Soc. Trans., 15, 603 (1987) J. Biol. Chem., 277, 26733 (2002) J. Biol. Chem., 278, 3466 (2003) Hepatology, 13, 683 (1991) Hepatology, 28, 944 (1998) Glycobiology, 11, 165, (2001) WO92 / 27303 WO02 / 31140

An object of the present invention is to provide an enzyme involved in sugar chain modification in which fucose at position 1 is α-linked to position 6 of N-acetylglucosamine at the reducing end of N-glycoside-linked complex type sugar chain (hereinafter, α1,
The present invention provides a mouse and its progeny whose genome has been modified such that the activity of 6-fucose modifying enzyme is reduced or deleted.
The mouse of the present invention and its progeny are useful for elucidating the physiological role of α1,6-fucose modifying enzyme and its relation to the pathological condition with the enzyme. It is also useful for the development of drugs targeting α1,6-fucose modifying enzymes.

The present invention relates to the following (1) to (6).
(1) The genome is reduced so that the activity of an enzyme involved in sugar chain modification in which the position 1 of fucose is α-linked to the position 6 of N-acetylglucosamine at the reducing end of N-glycoside-linked complex type sugar chain is reduced or deleted. A modified mouse or progeny thereof.
(2) The genomic gene of an enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the reducing end of N-glycoside-linked complex type sugar chain was knocked out, as described in (1) above. The mouse or progeny thereof.
(3) All the alleles on the genome of the enzyme involved in sugar chain modification in which the 1-position of fucose is α-linked to the 6-position of N-acetylglucosamine at the reducing end of N-glycoside-linked complex type sugar chain, The mouse or the progeny thereof according to the above (1) or (2).
(4) The enzyme involved in sugar chain modification in which position 1 of fucose is α-linked to position 6 of N-acetylglucosamine at the reducing end of N-glycoside-linked complex type sugar chain is α1,6-fucosyltransferase, The mouse according to any one of 1) to (3) or a progeny thereof.
(5) The mouse or progeny thereof according to (4), wherein the α1,6-fucosyltransferase is a protein encoded by a DNA selected from the following (a) or (b):
(a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2;
(b) a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having α1,6-fucosyltransferase activity.
(6) The mouse or progeny thereof according to (4), wherein the α1,6-fucosyltransferase is a protein selected from the group consisting of the following (a), (b) and (c).
(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1;
(b) a protein consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added in the amino acid sequence represented by SEQ ID NO: 1, and having α1,6-fucosyltransferase activity;
(c) a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having α1,6-fucosyltransferase activity;

According to the present invention, a mouse and its progeny whose genome has been modified such that the activity of α1,6-fucose modifying enzyme is reduced or deleted is provided.

The mouse or its progeny of the present invention includes any mouse or its progeny whose genome has been modified so that the activity of α1,6-fucose modifying enzyme is reduced or deleted, and any mouse or its progeny is included. .
In the present invention, any α1,6-fucose modifying enzyme may be used as long as it is an enzyme involved in a reaction in which the 6-position of N-acetylglucosamine at the N-glycoside-linked complex type sugar chain reducing end and the 1-position of fucose are α-linked. Enzymes are also included. Specific examples of the α1,6-fucose modifying enzyme include α1,6-fucosyltransferase.

In the present invention, α1,6-fucosyltransferase includes
A protein encoded by the DNA of the following (a) or (b),
(a) a DNA consisting of the base sequence represented by SEQ ID NO: 2
(b) DNA that hybridizes with a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and encodes a protein having α1,6-fucosyltransferase activity
Alternatively, the following proteins (c), (d) and (e) are mentioned.
(c) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1
(d) a protein comprising an amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted, inserted and / or added, and having α1,6-fucosyltransferase activity
(e) a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having α1,6-fucosyltransferase activity In the present invention, the protein hybridizes under stringent conditions DNA refers to, for example, a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like, using a DNA such as a DNA having the base sequence represented by SEQ ID NO: 2 or a fragment thereof as a probe. Means a DNA obtained by the method, specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M sodium chloride using a filter on which DNA derived from colonies or plaques was immobilized. Later, 0.1-2
DNA that can be identified by washing the filter with a double-concentration SSC solution (the composition of a single-concentration SSC solution is composed of 150 mM sodium chloride and 15 mM sodium citrate) at 65 ° C. can be mentioned. Hybridization is performed using Molecular Cloning, A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory Press, 1989 (hereinafter abbreviated as Molecular Cloning Second Edition), Current Protocols in Molecular Biology, John Wiley & Sons,
1987-1997 (hereinafter abbreviated as Current Protocols in Molecular Biology), DNA Cloning 1: Core Techniques, A Practical Approach, Second E
dition, Oxford University (1995) and the like. Specific examples of the hybridizable DNA include a DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 2, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more. As described above, particularly preferred are DNAs having a homology of 95% or more, most preferably 98% or more.

In the present invention, the amino acid sequence represented by SEQ ID NO: 1 comprises an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added, and α
Proteins having 1,6-fucosyltransferase activity are described in Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Nucleic Acids Research, 10 , 6487 (1982), Proc. Natl. Acad. Sci. ., USA
, 79 , 6409 (1982), Gene, 34 , 315 (1985), Nucleic Acids Research, 13 , 443.
Natl. Acad. Sci USA, 82 , 488 (1985), etc., using a site-directed mutagenesis method, for example, to encode a protein having the amino acid sequence represented by SEQ ID NO: 1. Means a protein that can be obtained by introducing a site-specific mutation into DNA to be transformed. The number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number is not particularly limited. The number of amino acids to be deleted, substituted or added by a well-known technique such as the site-directed mutagenesis method described above. The number is as small as possible, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5.

Further, in the present invention, a protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having α1,6-fucosyltransferase activity,
BLAST [J. Mol. Biol., 215 , 403 (1990)] and FASTA [Methods in Enz.
ymology, 183 , 63 (1990)].
And at least 80%, preferably 85% or more, of the protein having the amino acid sequence of
% Or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.

In the present invention, the expression that the genome has been modified such that the activity of the α1,6-fucose modifying enzyme is reduced or deleted means that a mutation is introduced into the expression regulatory region of the gene so that the expression of the enzyme is reduced or deleted. Or introducing a mutation into the amino acid sequence of the gene so as to reduce the function of the enzyme. Introducing a mutation means modifying a nucleotide sequence such as deletion, substitution, insertion and / or addition to a nucleotide sequence on the genome, and knocking out completely suppressing the expression or function of the modified genomic gene. To do it. A specific example of knocking out a genomic gene is an example in which all or a part of a target gene has been deleted from the genome. As a method for obtaining such a mouse or its progeny, any method can be used as long as the desired genome can be modified. Specific examples include a method of gene disruption targeting an enzyme gene, a method of introducing a mutation into an enzyme, and a method of producing a cloned individual using a nucleus of a cell subjected to a desired genetic modification.

Hereinafter, methods for producing and using the mouse and its progeny of the present invention will be described in detail.
1. Method of Generating Mouse and Its Progeny of the Present Invention (1) Technique of Gene Disruption Targeting Enzyme Gene The mouse and its progeny of the present invention target the gene of α1,6-fucose modifying enzyme and perform gene disruption. Can be produced by using Specific examples of the α1,6-fucose modifying enzyme include α1,6-fucosyltransferase.

The method for gene disruption includes any method that can disrupt the gene of the target enzyme. Examples include homologous recombination, RD
O method, a method using a retrovirus, a method using a transposon, and the like. Hereinafter, these will be described specifically.
(A) Production of Mouse of the Present Invention and Its Progeny by Homologous Recombination Method The mouse of the present invention and its progeny target the gene of α1,6-fucose modifying enzyme and perform homologous recombination on the target gene on the chromosome. It can be produced by using and modifying.

Modification of the target gene on the chromosome can be performed by the Manipulating the Mouse Embryo A Laborato
ry Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (
Hereafter abbreviated as "Manipulating the Mouse Embryo a Laboratory Manual"), Gene Targeting, A Practical Approach, IRL Press at Oxford
University Press (1993), Biomanual Series 8, Gene Targeting, Production of Mutant Mice Using ES Cells, Yodosha (1995) (hereinafter abbreviated as "Generation of Mutant Mice Using ES Cells"), etc. Using chromosome engineering techniques, for example, it can be performed as follows.

Obtain cDNA of α1,6-fucose modifying enzyme.
Based on the nucleotide sequence of the obtained cDNA, the genome DN of α1,6-fucose modifying enzyme
Prepare A.
Based on the base sequence of the genomic DNA, the target gene to be modified (for example, α1,6-
A target vector for homologous recombination of a structural gene of a fucose modifying enzyme or a promoter gene is prepared.

The prepared target vector is introduced into embryonic stem cells, and cells that have undergone homologous recombination between the target gene and the target vector are selected.
A germline chimera is selected by introducing the selected embryonic stem cells into a fertilized egg and transplanting the introduced fertilized egg into the oviduct or uterus of a pseudopregnant female mouse according to a known injection chimera method or a collective chimera method.

The selected target germline chimeras were bred, and from the offspring, an individual having a chromosome into which the introduced target vector was inserted by homologous recombination with the gene region on the genome encoding the α1,6-fucose modifying enzyme. select.
The selected individuals are bred together, and from the offspring, on both homologous chromosomes,
A homozygote having a chromosome inserted by the homologous recombination of the introduced target vector with the gene region on the genome encoding the α1,6-fucose modifying enzyme is selected.

By crossing the obtained homozygotes and obtaining their progeny, the mouse of the present invention and its progeny can be produced.
Examples of a method for obtaining cDNA and genomic DNA of α1,6-fucose modifying enzyme include the following methods.
Preparation of cDNA Total RNA or mRNA is prepared from mouse cells to be modified.

A cDNA library is prepared from the prepared total RNA or mRNA.
Based on the known amino acid sequence of the α1,6-fucose modifying enzyme, for example, a human amino acid sequence, a degenerative primer was prepared, and the α1,6-fucose modifying enzyme was prepared by PCR using the prepared cDNA library as a template. Obtain the gene fragment to be coded.

Using the obtained gene fragment as a probe, a cDNA library can be screened to obtain a cDNA encoding α1,6-fucose modifying enzyme.
As the mRNA of mouse cells, commercially available one (for example, Clontech) may be used, or total RNA may be prepared as follows, and mRNA may be prepared therefrom. As a method for preparing total RNA from cells, a guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, 1
54 , 3 (1987)], guanidine acid thiocyanate / phenol / chloroform (A
GPC) method [Analytical Biochemistry,
162 , 156 (1987); Experimental Medicine, 9 , 1937 (1991)].

Examples of a method for preparing mRNA as poly (A) ⁺ RNA from total RNA include an oligo (dT) -immobilized cellulose column method (Molecular Cloning, 2nd edition) and the like.
Furthermore, Fast Track mRNA Isolation Kit (Invitrogen), Quick Prep mRNA
MRNA can be prepared by using a kit such as Purification Kit (Pharmacia).

A cDNA library is prepared from the prepared mouse cell mRNA. cD
Methods for preparing NA libraries include Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, A Laboratory Manua
l, 2 nd Ed. (1989), or a commercially available kit such as SuperSc
ript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technol
ogies) and a method using a ZAP-cDNA Synthesis Kit (STRATAGENE).

As a cloning vector for preparing a cDNA library, any phage vector, plasmid vector, or the like can be used as long as it can autonomously replicate in Escherichia coli K12 strain. Specifically, ZAP Express [STRATAGENE, Strategies, 5 , 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17 , 9494 (1989)] , Lambd
a ZAP II (STRATAGENE), λgt10, λgt11 [DN Cloning
DNA cloning, A Practical Approach, 1 , 49
(1985)], λTriplEx (Clontech), λExCell (Pharmacia), pT7T318U (Ph
armacia), pcD2 [Molecular Cellular Biology (Mol. Cell. Biol.
), 3 , 280 (1983)] and pUC18 [Gene, 33 , 103 (1985)].

As the host microorganism, any microorganism can be used, but Escherichia coli is preferably used. Specifically, Escherichia coli XL1-Blue MRF '[ST
RATAGENE, Strategies, 5 , 81 (1992)], Escherichia coli
C600 [Genetics, 39 , 440 (1954)], Escherichia coli Y108
8 [Science, 222 , 778 (1983)], Escherichia coli Y1090 [Science, 222 , 778 (1983)], Escherichia coli NM522 [Journal of Molecular Biology (J. Mol. Biol) .), 166 , 1 (1983)], Escheric
hia coli K802 [Journal of Molecular Biology (J. Mol. Biol
.), 16 , 118 (1966)] and Escherichia coli JM105 [Gene, 38 , 275].
(1985)].

This cDNA library may be used for subsequent analysis as it is, but in order to reduce the proportion of incomplete cDNA and to obtain full-length cDNA as efficiently as possible,
Oligocap method developed by Sugano et al. [Gene, 138 , 171 (1994);
Gene), 200 , 149 (1997); Protein nucleic acid enzyme, 41 , 603 (1996); Experimental medicine, 11 , 24
91 (1993); cDNA cloning (Yodosha) (1996); Gene library preparation method (Yodosha) (1994)] may be used for the following analysis.

On the basis of the amino acid sequence of the α1,6-fucose modifying enzyme, a degenerative primer specific to the base sequence at the 5 ′ end and 3 ′ end of the base sequence predicted to encode the amino acid sequence is prepared, PCR method using the prepared cDNA library as a template [PCR Protocols, Academic Pr.
ess (1990)], a gene fragment encoding α1,6-fucose modifying enzyme can be obtained.

The fact that the obtained gene fragment is a DNA encoding an α1,6-fucose modifying enzyme can be determined by a commonly used nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. [Proceedings of the National Academy. Of Science (P
roc. Natl. Acad. Sci. USA), 74 , 5463 (1977)] or ABIPRISM.
It can be confirmed by analysis using a base sequence analyzer such as 377 DNA sequencer (manufactured by PE Biosystems).

Using this gene fragment as a DNA probe, colony hybridization or plaque hybridization (molecular cloning second edition) is performed on cDNA or a cDNA library synthesized from mRNA contained in mouse cells to be modified to obtain α1. , 6-fucose modifying enzyme DNA can be obtained.

In addition, using the primers used to obtain the gene fragment encoding the α1,6-fucose modifying enzyme, PCR was performed using a cDNA or cDNA library synthesized from mRNA contained in mouse cells to be modified as a template. By performing screening using, DNA of α1,6-fucose modifying enzyme can also be obtained.

The base sequence of the DNA encoding the obtained α1,6-fucose modifying enzyme is
A commonly used nucleotide sequence analysis method, for example, the dideoxy method of Sanger et al. [Proceedings of the National Academy of Sciences (Proc.
Natl. Acad. Sci. USA), 74 , 5463 (1977)] or ABIPRISM37.
The base sequence of the DNA is determined by analysis using a base sequence analyzer such as 7 DNA sequencer (manufactured by PE Biosystems).

Based on the base sequence of the determined cDNA, a base sequence database such as GenBank, EMBL and DDBJ is searched by using a homology search program such as BLAST, thereby obtaining α1,6-fucose among the genes in the database. The gene encoding the modifying enzyme can also be determined.
The nucleotide sequence of the gene encoding the α1,6-fucose modifying enzyme obtained by the above method includes, for example, the nucleotide sequence of SEQ ID NO: 2.

Based on the determined nucleotide sequence of the DNA, the cDNA of α1,6-fucose modifying enzyme was chemically synthesized by a DNA synthesizer such as a DNA synthesizer model 392 manufactured by Perkin-Elmer using the phosphoramidite method. You can also get it.
Examples of a method for preparing a genomic DNA of the α1,6-fucose modifying enzyme include the following methods.

Methods for preparing genomic DNA Examples of methods for preparing genomic DNA include known methods described in Molecular Cloning Second Edition, Current Protocols in Molecular Biology, and the like. In addition, genomic DNA of α1,6-fucose modifying enzyme can be isolated by using a genomic DNA library screening system (Genome Systems) or Universal GenomeWalker ^™ Kits (CLONTECH).

The nucleotide sequence of the genomic DNA of the α1,6-fucose modifying enzyme obtained by the above method is
It can be confirmed from the fact that it contains the cDNA sequence of the α1,6-fucose modifying enzyme obtained by the above method.
The target vector for homologous recombination of the target gene is Gene Targeting,
A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8, Gene Targeting, Generation of Mutant Mouse Using ES Cells (Yodosha) (1995), etc. . As the target vector, any of a replacement type, an insertion type, and a gene trap type can be used.

As a method for introducing a target vector into embryonic stem cells, any method for introducing DNA into animal cells can be used. For example, electroporation [Cytotechnology, 3 , 133 (1990)] ], Calcium phosphate method [Japanese Unexamined Patent Application Publication No. 2-227075], lipofection method [Proceedings of
The National Academy of Sciences (Proc. Natl. Acad. Sci. USA),
84 , 7413 (1987)], the injection method [Manipulating the Mouse Embryo AL
aboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1
994) (hereinafter abbreviated as Manipulating Mouse Embryo 2nd Edition)], a method using a particle gun (gene gun) [Patent No. 2606856, Patent No. 2517813],
DEAE-dextran method [Bio Manual Series 4-Gene transfer and expression
Analytical method (Yodosha), Takashi Yokota, Ken-ichi Arai (1994)], virus vector method [manipulating mouse embryo 2nd edition], and the like.

As a method for efficiently selecting homologous recombinants, for example, Gene Targeting, A Pr
positive selection, promoter selection, negative selection, etc. described in actical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Production of Mutant Mice Using ES Cells (Yodosha) (1995), etc. A method such as poly A selection can be used. Specifically, in the case of a target vector containing the hprt gene, after introduction into an embryonic stem cell lacking the hprt gene,
Embryonic stem cells are cultured in a medium containing aminopterin, hypoxanthine and thymidine, and aminopterin-resistant strains are selected, whereby positive selection for selecting homologous recombinants containing the hprt gene can be performed. In the case of a target vector containing a neomycin resistance gene, the embryonic stem cells into which the vector has been introduced are cultured in a medium containing G418, and G418-resistant strains are selected to select homologous recombinants containing the neomycin resistance gene. You can make a selection. In the case of a target vector containing the DT gene, the embryonic stem cells into which the vector has been introduced are cultured,
The strain that has grown is selected (the recombinant inserted randomly into the chromosome other than homologous recombination cannot grow due to DT toxicity because the DT gene is integrated into the chromosome and expressed). Negative selection to select for homologous recombinants that do not contain can be performed. Methods for selecting a desired homologous recombinant from the selected cell lines include Southern hybridization (genetic DNA cloning, 2nd edition) for genomic DNA and PCR [PCR Protocols, Academic Press (1990)].

When embryonic stem cells are incorporated into a fertilized egg using the collective chimera method, generally 8
It is preferable to use a fertilized egg at a developmental stage before the cell stage. When embryonic stem cells are incorporated into a fertilized egg using the injection chimera method, it is generally preferable to use a fertilized egg in the stage of blastocyst development from the 8-cell stage.
When a fertilized egg is transplanted into a female mouse, the fertilized egg obtained in a pseudopregnant female mouse in which fertilization has been induced is artificially transplanted and implanted by mating with a vasectomized male non-human mammal. Pseudopregnant female mice are preferably obtained by natural mating. After administration of luteinizing hormone-releasing hormone (hereinafter abbreviated as LHRH) or an analog thereof, fertilization is achieved by mating with male mice. Induced pseudopregnant female mice can also be obtained. As analogs of LHRH, for example, [3,5-Dil-Tyr5]
-LHRH, [Gln8] -LHRH, [D-Ala6] -LHRH, des-Gly10- [D-His (Bzl) 6] -LHRH ethylami
de and the like.
(b) Preparation of the mouse and its progeny of the present invention by the RDO method The mouse and its progeny of the present invention target the α1,6-fucose modifying enzyme gene and use the RDO (RNA-DNA oligonucleotide) method, for example, It can be manufactured as follows.

As described above, cDNA or genomic DNA of α1,6-fucose modifying enzyme
Is prepared, and the base sequence of the prepared cDNA or genomic DNA is determined.
Based on the determined DNA sequence, a portion encoding α1,6-fucose modifying enzyme,
An RDO construct of an appropriate length including the untranslated region portion or the intron portion is designed and synthesized.

The synthesized RDO is introduced into embryonic stem cells, and embryonic stem cells in which a mutation has occurred in the targeted enzyme, ie, the α1,6-fucose modifying enzyme, are selected.
The selected embryonic stem cells are introduced into a fertilized egg according to the injection chimera method or the collective chimera method, and the introduced fertilized egg is transplanted into the oviduct or uterus of a pseudopregnant female mouse to select a germline chimera.

After breeding the selected germline chimera and breeding the offspring, an individual with a chromosome into which the introduced target vector has been inserted by homologous recombination with the gene region on the genome encoding α1,6-fucose modifying enzyme select.
The selected individuals are bred together, and from the offspring, on both homologous chromosomes,
A homozygote having a chromosome inserted by the homologous recombination of the introduced target vector with the gene region on the genome encoding the enzyme involved in the α1,6-fucose modifying enzyme is selected.

By crossing the obtained homozygotes and obtaining their progeny, the mouse of the present invention and its progeny can be produced.
For the introduction of RDO into embryonic stem cells, the method for introducing a target vector described in the above 1 (1) (a) can be used.
RDO can be prepared by a conventional method or by using a DNA synthesizer.

Methods for introducing RDO into embryonic stem cells and selecting cells in which the gene for the α1,6-fucose modifying enzyme has been mutated include Molecular Cloning, Second Edition, Current
A method for directly detecting a mutation of a gene on a chromosome described in Protocols in Molecular Biology and the like can be mentioned.
The RDO construct is described in Science, 273 , 1386, (1996); Nature Medicine, 4 , 285, (1998); Hepatology.
logy), 25 , 1462, (1997); Gene Therapy, 5 , 1960, (1999)
; Gene Therapy, 5 , 1960, (1999); Journal of Molecular Medicine (J. Mol. Med.), 75 , 829, (1997); Proceedings of the National. Academy of Science (Proc. Natl. Acad. Sci.
USA), 96 , 8774, (1999); Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 96 , 8768, (1999); Nucleic Acid Research. (Nuc. Acids. Res.), 27 , 1323, (1999); Invest.Dematol., 111 , 1172, (1998);
Nature Biotech., 16 , 1343, (1998); Nature Biotech., 18 , 43, (2000); Nature Biotech., 18 , 555, (2000).
(C) Production of the mouse of the present invention and its progeny by a method using a transposon The mouse of the present invention and its progeny were obtained from Nature Genet.
, 25 , 35, (2000), etc., and by selecting a mutant of the α1,6-fucose modifying enzyme.

The transposon system is a system in which a foreign gene is randomly inserted into a chromosome to induce a mutation.Usually, a foreign gene inserted in the transposon is used as a vector to induce a mutation, and this gene is used as a chromosome. A transposase expression vector for random insertion above is simultaneously introduced into the cells.

Any transposase can be used as long as it is suitable for the sequence of the transposon to be used.
As the foreign gene, any gene can be used so long as it can induce mutation in the DNA of a cell.
For introducing a gene into a cell, the method for introducing a target vector described in the above 1 (1) (a) can be used.
(2) Technique for Introducing Mutation in Enzyme The mouse of the present invention and its progeny are obtained by introducing a mutation in the gene of the α1,6-fucose modifying enzyme and selecting a desired mouse in which the enzyme has been mutated. It can be manufactured by using the technique described below.

Specifically, a desired mouse in which a gene of α1,6-fucose modifying enzyme has been mutated from a mutant born from a germ cell treated by the mutagenesis treatment or a spontaneously generated mutant is obtained. There is a selection method.
Germ cells include cells having the ability to form individuals, such as sperm, eggs, and embryonic stem cells.

As the mutagenesis treatment, any treatment can be used as long as it induces a point mutation, deletion or frameshift mutation in the DNA of the cells.
Specific examples include treatment with ethylnitrosourea, nitrosoguanidine, benzopyrene, and acridine dye, irradiation with radiation, and the like. Various alkylating agents and carcinogens can also be used as mutagens. Examples of a method for causing a mutagen to act on cells include, for example, tissue culture technology, 3rd edition (Asakura Shoten), edited by the Japanese Society for Tissue Culture (1996), Nature Genetics,
24 , 314, (2000) and the like.

Spontaneously occurring mutants include those that occur spontaneously by continued normal breeding without special mutagenesis treatments.
(3) Method for producing a cloned individual using the nucleus of a cell subjected to the intended genetic modification The mouse of the present invention and its progeny are cloned mice (T. Wakay) described in the literature.
ama et al .; Nature, 394 , 369, 1998; T. Wakayama et al .; Nature Genetics, 22 , 127,
For example, it can be produced as follows using the production method of (1999).

The above 1. Using the method described in (1) or (2), a mutation is introduced into the α1,6-fucose modifying enzyme gene on the chromosome of any mouse cell.
Next, the nucleus of the obtained cell is initialized (operation for returning the nucleus to a state in which generation can be repeated again).
Development is initiated by injecting nuclei of reprogrammed cells into unfertilized eggs of enucleated mice.

By artificially transplanting and implanting eggs that have started to develop into female mice,
A heterozygote in which a mutation has been introduced into the α1,6-fucose modifying enzyme gene is obtained.
By crossing the obtained heterozygotes, homozygotes are obtained.
By crossing the obtained homozygotes and obtaining their progeny, the mouse of the present invention and its progeny can be produced.

It is known that the method of reprogramming the nucleus of cells differs depending on the type of non-human mammal, but in the case of mice and the like, exogenous non-human mammals of non-human mammal The nucleus of the cell into which the gene has been introduced is injected for several hours,
It is preferable to initialize by culturing for 6 hours.
It is also known that the method of initiating development of reprogrammed nuclei in enucleated unfertilized eggs varies depending on the type of non-human mammal, but in the case of mice, etc., foreign genes are introduced. Unfertilized eggs into which the nuclei of the transformed cells have been injected are stimulated with an egg activating substance (eg, strontium) and treated with a cell division inhibitor (eg, cytochalasin B) to suppress the release of the second polar body It is preferable to start the generation.

As a method of artificially transplanting and implanting eggs that have started to develop into female mice,
The above 1. (1) (a).
2. Use of the mouse of the present invention and its progeny (1) Analysis of the physiological function of α1,6-fucose modifying enzyme using the mouse of the present invention and its progeny Since the genome is modified so that the activity of the enzyme is reduced or deleted, 1) the physiological role of the enzyme in the developmental process, and 2) the physiological role of the enzyme in the process from development to adulthood. Role, 3)
It is possible to investigate the physiological role of the enzyme in adults.
As an α1,6-fucose modifying enzyme, α1,6-fucosyltransferase is known, but it can be determined at the level of various organs whether an isozyme having the same enzyme activity is present.
In addition, by comparing normal individuals, heterozygotes, and homozygotes, it is possible to observe the physiological effects of a quantitative change in α1,6-fucose modifying enzyme.
(2) Method of pharmacological evaluation of substance using mouse or its progeny of the present invention The mouse or its progeny of the present invention has a causal relationship with a disease suspected of being associated with α1,6-fucose modifying enzyme. It is useful as a tool to clarify relationships and find coping or radical therapies.

Specifically, a test substance is administered to a mouse of the present invention or a progeny thereof, and various physical parameters such as blood pressure, respiratory rate, and body weight of the animal are measured and compared with an animal to which the test substance is not administered. The pharmacological evaluation of the test substance can be carried out by examining pharmacological actions such as observation of behavior and behavior, and histopathological examination. At times, knowledge of symptoms similar to those observed in human disease is important and can provide important data for therapeutic drug development.

Further, a disease model animal in which various diseases are induced in the mouse of the present invention and its progeny is prepared, a test substance is administered to the disease model animal, and various blood pressure, respiratory rate, body weight, etc. of the disease model animal are obtained. Measurement of physical parameters, observation of pathology, appearance, behavior,
By performing histopathological examination and the like in comparison with the pathological model animal to which the test substance is not administered, the test substance in the animal in which the activity of the α1,6-fucose modifying enzyme is reduced or deleted, Pharmacological evaluation of efficacy and side effects can be performed. Further, based on this evaluation, a substance that is preferable as a therapeutic drug for the disease can be selected.

Examples of the disease induced in the mouse of the present invention and its progeny include heart disease (eg, acute heart failure, chronic heart failure, myocarditis, etc.), respiratory disease, joint disease (eg, rheumatoid arthritis, osteoarthritis, etc.), Kidney disease (eg, renal failure, glomerulonephritis, Ig
A nephropathy), arteriosclerosis, psoriasis, hyperlipidemia, allergic diseases (eg, asthma, allergic rhinitis, atopic dermatitis, etc.), bone diseases (eg, osteoporosis, rickets, osteomalacia, Hypocalcemia), blood diseases, cerebrovascular injuries, traumatic encephalopathy, infectious diseases, dementia, cancer, diabetes, liver diseases, skin diseases, neurodegenerative diseases and chronic inflammatory diseases. Preparation of the disease state model animal is `` manual disease model mouse '' (Molecular Medicine, 31 extra edition, Nakayama Shoten (1994)),
"Animal disease model for illustration and pharmacology" (Nishimura Shoten (1984)), "Arthritis model animal" (Dental medicine publication (1985)), "Neuro-muscular disease model animal" (Dental medicine publication)
(1982)], “Reactive oxygen and pathological conditions From disease model to bedside” [Academic Publishing Center (1992)] and the like.
(3) Method of pharmacological evaluation of substance using cells obtained from mouse and its progeny of the present invention Various cells obtained from mouse and its progeny of the present invention are brought into contact with a test substance, By examining pharmacological effects such as various cellular responses such as an increase in intracellular Ca ²⁺ concentration and changes in cell morphology, it is possible to perform pharmacological evaluation of test substances on the cells in comparison with cells of the same type. it can.

In addition, various types of cells can be obtained by inducing differentiation of embryonic stem cells obtained from the mouse of the present invention and progeny thereof. As a method for inducing differentiation, a method of inducing a teratoma (teratoma) mixed with various tissues by implanting embryonic stem cells subcutaneously into an animal of the same type and same strain (manipulating mouse embryo 2nd edition) ), By culturing in vitro under appropriate conditions, endoderm cells, ectoderm cells, mesoderm cells, blood cells, endothelial cells, chondrocytes, skeletal muscle cells, smooth muscle cells, cardiomyocytes, nerve cells, glial cells, Methods for inducing differentiation into epithelial cells, melanocytes, and keratinocytes (Reprod. Fertil. Dev., 10 , 31, 1998) can be mentioned. The differentiated cells are contacted with a test substance, and various cell responses such as an increase in intracellular Ca ²⁺ concentration and changes in cell morphology are compared with cells in the absence of the test substance. By examining the pharmacological action of, pharmacological evaluation of the test substance on the cells can be performed. According to these methods, pharmacological evaluation can be performed on cells that are difficult to remove from a living body of a human patient or cells that exist only in a small number.
(4) Preparation of Genetically Modified Animal Using Embryonic Stem Cell, Egg, Sperm and Nucleus of Mouse and Progeny of the Present Invention Using embryonic stem cell, egg, sperm or nucleus obtained from mouse of the present invention and its progeny And 1. According to the method described in (1), it is possible to obtain a genetically modified mouse in which the genome is modified so that the activity of the α1,6-fucose modifying enzyme is reduced or deleted, and the gene on the chromosome is further modified. In particular, a gene known to cause a disease state, which is the disruption of the function of the gene, as described in 1. above. Knockout mice deficient using the homologous recombination technique described in (1) and transgenic mice in which a dominant negative body gene that inhibits the function of the gene is introduced and expressed are useful as disease state animal models.
(5) Production of a genetically modified animal by crossing the mouse of the present invention or its progeny with an animal of the same or different strain and use of the produced genetically modified animal An animal of the same or different strain as the mouse of the present invention or its progeny (for example, , A human disease model animal), the genome is modified so that the activity of α1,6-fucose modifying enzyme is reduced or deleted, and a certain phenotype (for example, a symptom similar to a human disease state) is introduced. The genetically modified mouse shown in the figure can be obtained. If the mouse to be bred is a human disease model animal, a pathological model animal whose genome is modified so that the activity of α1,6-fucose modifying enzyme is reduced or deleted, and which exhibits a symptom similar to a human pathological condition as an expression system Is obtained. Methods of mating include in vitro fertilization in addition to natural mating. As the disease model animal, any disease model animal can be used, regardless of whether the disease is congenital or acquired. For example, an acquired pathological model animal is "manual disease model mouse" [Molecular Medicine, 31 extra edition, Nakayama Shoten
(1994)), `` Pathological animal models for illustration and pharmacology '' (Nishimura Shoten (1984)),
"Arthritis model animals" (Imedicine Publishing (1985)), "Nerve and muscle disease model animals" [
Toyoyaku Shuppan (1982)], "Reactive Oxygen and Pathological Conditions From Disease Model to Bedside" [
Academic Press Center (1992)].
(6) Pharmacological evaluation method of a substance using a genetically modified animal A test substance is administered to the genetically modified disease model animal obtained by the method described in the above (4) and (5), and the blood pressure and respiration of the disease model animal are administered. Measurement of various physical parameters such as number, body weight, etc., disease state, appearance, behavioral observation, histopathological examination, etc. are performed in comparison with the disease state model animal to which the test substance is not administered, whereby the test substance Pharmacological evaluation such as efficacy and side effects for the disease. Further, based on this evaluation, a substance that is preferable as a therapeutic drug for the disease state can be selected.

The present invention will be described more specifically with reference to the following examples, which are merely illustrative of the present invention and do not limit the scope of the present invention.

Embodiment 1 FIG. Generation of transgenic mice lacking both α1,6-fucosyltransferase alleles Generation of mice lacking the genomic region containing the translation initiation codon of both α1,6-fucosyltransferase (hereinafter also referred to as FUT8) alleles This was performed as follows.

1. Isolation of genomic region including mouse α1,6-fucosyltransferase (FUT8) gene translation initiation codon Full length of pig FUT8 cDNA [Journal of Biological Chemistry (J. B
iol. Chem., 271 , 27810 (1996)], a fragment (412 bp) containing the 5'-terminal untranslated region 39 bp to the translated region 373 bp was prepared by digestion with the restriction enzyme SacI. Using this as a probe, Molecular Cloning, 2nd edition [Molecular Cloning, A Laborat
ory Manual, 2 nd Ed. (1989)], a 13.9 Kb genomic clone containing an exon in which the mouse FUT8 translation initiation codon is located was isolated from a 129SVJ strain mouse l-phage genomic library (STRATEGENE). (FIG. 1).

Next, the obtained genomic clone was digested with various restriction enzymes,
Molecular cloning 2nd edition [Molecular Cloning, A Laboratory Manual, 2 nd Ed. (1989)] according to a known method. Encodes the exon and upstream intron region of the translation initiation codon among the positive restriction enzyme fragments
An XbaI-XbaI fragment (about 2.9 Kb) and a SacI-SacI fragment (about 6.6 Kb) encoding an exon and a downstream intron region located at the translation initiation codon were selected, and pBluescriptII was selected.
Each was inserted into KS (+) (Strategene) (FIG. 1).

2. Construction of a targeting vector plasmid for disrupting the mouse α1,6-fucosyltransferase FUT8) gene translation initiation codon In the XbaI-XbaI region (about 2.9 Kb) obtained in Section 1 of the present example, XbaI at the 5′-terminal side -SacI
The region (2.6 Kb) was used as the 5'-terminal homologous region, while the SacI-SacI obtained in section 1 of this example was used.
Of the region (about 6.6 Kb), by arranging the 3 'terminal HindIII-XhoI region (about 6.1 Kb) as the 3' terminal homologous region, the SacI-HindIII region containing the translation initiation codon
(184 bp) was deleted to construct a targeting vector plasmid. The details are shown below.

First, plasmid pMC1DTpA [Transgenic Research (Transgenic Research)
arch), 8 , 215 (1999)], digested with restriction enzymes XhoI and NotI, and ligated to a 1.5 Kb fragment containing the obtained diphtheria toxin A chain (DT-A) gene with a NotI-XhoI adapter. Both ends were replaced with XhoI recognition sites. On the other hand, FUT8 obtained in the first section of this example
Restriction enzyme X for pBluescriptII KS (+) containing 3'-terminal SacI-SacI region (about 6.6 Kb)
By reacting with hoI, an 8.3 Kb fragment containing the HindIII-XhoI region (about 6.1 Kb) was obtained. The XhoI-XhoI fragment (8.3 Kb) containing the FUT8 3′-terminal homologous region obtained above and the Xho-Xho fragment (1.5 Kb) containing the DT-A gene were ligated to construct plasmid I.

Next, pGT1.8IresBgeo [Proceedings of National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 91 , 4303 (1994)]
After complete digestion with the restriction enzyme SalI, further partial digestion with the restriction enzyme SacI, the internal ribosome translation initiation site (IRES), β-galactosidase gene (LacZ),
A 4.9 Kb fragment containing an expression unit for drug selection (hereinafter referred to as an IRES-LacZ-Neo ^r -pA cassette) to which a neomycin resistance gene (Neo ^r ) and a polyA addition signal (pA) were linked was obtained. On the other hand, the XbaI-XbaI region (about 2.9 Kb) of the FUT8 5 ′ terminal side obtained in the first section of this example
With pBluescriptII KS (+) containing restriction enzymes SacI and SalI to give XbaI-
A 5.3 Kb fragment containing the SacI region (2.6 Kb) was obtained. SacI-Sa containing the SacI-SalI fragment (5.3 Kb) containing the FUT8 5'-terminal homology region obtained above and the IRES-LacZ-Neo ^r -pA cassette
The lI fragment (4.9 Kb) was ligated to construct plasmid II.

Finally, a 7.6 Kb fragment obtained by digesting plasmid II with the restriction enzymes NotI and SalI was ligated to a 9.4 Kb fragment obtained by digesting plasmid I with the restriction enzymes NotI and SalI, thereby obtaining the mouse FUT8 gene. A targeting vector plasmid for disruption (17.0 Kb) was constructed (FIG. 2).
3. Homologous recombination of α1,6-fucosyltransferase (FUT8) gene genomic region in mouse embryonic stem cells (1) Preparation of feeder cells Mouse embryonic stem cells used for homologous recombination use mouse primary fibroblasts (EMFI cells) as feeders. The culture was used and maintained. The feeder cells used for maintaining the culture of embryonic stem cells were prepared according to the following description.

First, Gene targeting [Gene Targeting, IRL PRESS at Oxford Univ.
As described in ersity Press (1993)], fetuses were excised from 13.5 to 15.5 days after fertilization from female neomycin-resistant transgenic mice aged 8 weeks or older (provided by Prof. Masaru Okabe, Osaka University Genetic Information Center). Using this as a material, primary fibroblasts (EM
After preparing FI cells, the proliferation ability was inactivated by mitomycin C treatment. Subsequently, the mitomycin-treated EMFI cells were placed in an FM medium [10% fetal calf serum (Invitroge
n), 55 μmol / l β-mercaptoethanol (Invitrogen), 1 mmol / l MEM sodium pyruvate (Invitrogen), 0.1 mmol / l MEM non-essential amino acid (Invitrogen)
gen), 3 mmol / l adenosine (SIGMA), 3 mmol / l guanosine (SIGMA), 3
mmol / l cytidine (SIGMA), 3 mmol / l uridine (SIGMA), 1 mmol / l thymidine (SIGMA), 2 mmol / l L-glutamine (Invitrogen), 100 units / ml penicillin and 100 μg / ml streptomycin (Invitrogen) In a Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 2.5 × 10 ⁵ cells / ml, and then 0.1% gelatin-coated cell culture dishes (6 cm diameter, 10 cm diameter; Asahi Seeds on a flat bottom plate (96-well, 24-well, 6-well; Asahi Technograss) coated with 0.1% gelatin and treated with 0.1% gelatin and cultured for 24 hours at 5% CO ₂ at 37 ° C. A plate was made. The prepared feeder plate was used within one week after the preparation. As the feeder cells, besides using the above-mentioned feeder cells, for example, Neo-resistant primary cultured cells (catalog number YE9284100) commercially available from Lifetech Oriental Co., Ltd. can also be used.

(2) Introduction of targeting vector into embryonic stem cells Mouse embryonic stem cells D3 (provided by Professor Takashi Muramatsu, Nagoya University School of Medicine, and maintained at the Osaka University Genetic Information Center) were cultured using ESM medium [20% fetal bovine embryo Serum (In
vitrogen), 55 μmol / l β-mercaptoethanol (Invitrogen), 1 mmol / l
MEM sodium pyruvate (Invitrogen), 0.1 mmol / l MEM non-essential amino acid (I
nvitrogen), 3 mmol / l adenosine (SIGMA), 3 mmol / l guanosine (SIGMA)
3mmol / l Cytidine (SIGMA), 3mmol / l Uridine (SIGMA), 1mmol / l
Thymidine (SIGMA), 2 mmol / l L-glutamine (Invitrogen), 100 units / ml penicillin and 100 μg / ml streptomycin (Invitrogen), 1000 units / ml
Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing ESGRO ^™ (mouse recombinant leukemia inhibitory factor; SIGMA Invitrogen)]. First, freeze-thawed D3p11 cells were seeded on a feeder plate prepared using a 6 cm diameter dish, and cultured under conditions of 5% CO ₂ and 37 ° C. Twenty-four hours later, D3 cells were subcultured to three feeder plates prepared using a 10 cm diameter dish.

The introduction of the targeting vector plasmid obtained in Section 2 of this Example into D3 cells was carried out by electroporation [Cytotechnology, 3 , 1
33 (1990)]. First, 20 μg of the targeting vector plasmid was linearized by digestion with the restriction enzyme NotI, and then phenol /
Chloroform extraction and ethanol precipitation were performed to obtain a 1 μg / μl solution. On the other hand, the culture supernatant was removed from D3p11 cells 48 hours after subculture on a 10 cm diameter dish, and replaced with fresh ESM medium. After confirming that the D3 cells reached 70% confluence, they were suspended in a PBS buffer (Invitrogen) to 1 × 10 ⁷ cells / ml. Cell suspension
After mixing 800 μl (8.0 × 10 ⁶ ) with 20 μg of the above linearized plasmid, cell-DNA
Transfer the entire volume of the mixture to a Gene Pulser Cuvette (inter-electrode distance 4 mm) (BIO-RAD), and use a cell fusion device Gene Pulser (BIO-RAD) to introduce the gene under the conditions of a pulse voltage of 250 V and an electric capacity of 500 μF. went. After the introduction, the cell suspension was suspended in 40 ml of ESM medium, and seeded on three feeder plates prepared using a 10 cm diameter dish and two feeder plates prepared using a 6 cm diameter dish. 16 under the condition of 5% CO ₂ and 37 ° C
After culturing for more than an hour, the culture supernatant was removed and replaced with an ESM medium supplemented with 150 μg / ml G418 (Invitrogen). This medium exchange operation was repeated almost every day for 8 days of culture to obtain drug-resistant strains. As mouse embryonic stem cells, in addition to the mouse embryonic stem cells described above, embryonic stem cells derived from 129 strain mouse, for example, AT
Mouse embryonic stem cell D3 (CRL-11632) available from CC, Cell & Technolo, USA
Embryonic stem cells derived from 129 strain mice produced by gies and DNX Transgenic Sciences can also be used.

(3) Acquisition of targeting vector-introduced strain An arbitrary 343 colonies were collected from the drug-resistant strain obtained in (2) of this section by the following procedure.
The culture supernatant was removed from the dish in which the drug-resistant clone appeared, and a phosphate buffer solution was injected, followed by transfer under a stereoscopic microscope. Next, the colonies were scraped off using a pipetman (GILSON), sucked, and collected into a round-bottom 96-well plate. After trypsinization, each clone was seeded on a feeder plate prepared using a flat-bottomed 24-well plate, and cultured using an ESM medium until confluence was achieved. After the culture, each clone on the plate was trypsinized to a total volume of 350 μl. Of these, 300 μl was mixed with an equal amount of a freezing medium [20% DMSO, 80% FM medium] to prepare a master plate, which was subjected to cryopreservation. The remaining 50 μl of the cell suspension was seeded on a gelatin-coated flat-bottomed 24-well plate (Asahi Techno Glass Co., Ltd.) to form a replica plate.
The cells were cultured using M medium until the cells became confluent.

(4) Diagnosis of homologous recombination by genome southern blot using FUT8 genomic region 5'-terminal probe Genome Southern using 5'-terminal probe in the following procedure for 343 clones obtained in this section (3) Diagnosis of homologous recombination was performed by blotting.
First, genomic DNA of each clone was prepared from the replica plate prepared in this section (3) according to a known method [Nucleic Acids Research (Nucleic Acids Research), 3 , 2303, (1976)], and a TE buffer ( pH 8.0) (10 mmol / l Tris-HCl, 1 mmo
l / l EDTA].

On the other hand, the genomic clone containing the FUT8 translation initiation codon (13
.9 Kb), a fragment approximately 500 bp upstream from the restriction enzyme XbaI recognition site at the 5 ′ end was prepared (FIG. 2).
After digesting the genomic DNA with the restriction enzyme PstI, molecular cloning 2nd edition [Molecular Cloning, AL] was performed using the above FUT8 5'-end fragment (500 bp) as a probe.
aboratory Manual, 2 nd Ed. (1989)] according to a known method.

Treatment with the restriction enzyme PstI generated a DNA fragment of about 7.5 Kb from the wild-type FUT8 allele. On the other hand, by the same restriction enzyme treatment, a DNA fragment of about 11.0 Kb was generated from an allele in which homologous recombination with the targeting vector had occurred (FIG. 2).
According to this method, about 7.5 Kb and about 11
A specific fragment of .0 Kb was found. Since the quantitative ratio of both fragments was 1: 1, it was confirmed that this clone was a homologous recombinant in which one FUT8 allele was replaced with a vector sequence.

(5) Diagnosis of homologous recombination by genome southern blot using FUT8 genomic region 3'-terminal probe Genome Southern using 3'-terminal probe in the following procedure for 343 clones obtained in this section (3) Diagnosis of homologous recombination was performed by blotting.
First, from the SacI-SacI region (about 6.3 Kb) obtained in the first section of this Example, a fragment containing a restriction enzyme EcoRV recognition site located about 500 bp upstream from the restriction enzyme SacI recognition site at the 3 ′ end was prepared. .

After digesting the genomic DNA prepared in this section (4) with the restriction enzyme SacI, the above FUT83
'Molecular cloning 2nd edition [M
olecular Cloning, A Laboratory Manual, 2 nd Ed. (1989)] according to a known method.
Treatment with the restriction enzyme SacI generated a DNA fragment of about 6.6 Kb from the wild-type FUT8 allele. On the other hand, a DNA fragment of about 8.6 Kb was generated from the allele that had undergone homologous recombination with the targeting vector by the same restriction enzyme treatment (FIG. 2).

According to this method, genomic DNA of 4 clones that showed positive in this section (4)
Specific fragments of .6 Kb and about 8.6 Kb were found. Since the quantitative ratio of both fragments was 1: 1, it was confirmed that this clone was a homologous recombinant in which one FUT8 allele was replaced with a vector sequence.
4. Acquisition of mouse in which α1,6-fucosyltransferase (FUT8) gene is disrupted (1) Acquisition of chimeric mouse using embryonic stem cells in which both copies of FUT8 allele are disrupted by one copy One of FUT8 established in section 3 of this example A known method [Manipulating the Mouse Embryo] was used for 4 clones of the embryonic stem cell in which the allele was disrupted.
(Manupirating the Mouse Embryo), Cold Spring Harbor laboratory Press (1
994)], three clones maintaining a normal karyotype were selected. Next, the Guide to Technuq in Mouse Development [Guide to Technuq
ues in Mouse Development, Methods in Enzymology, Volume 225, Academic Pr
ess (1993)], the three clones of embryonic stem cells described above were each microinjected into the blastocyst space obtained from a C57BL / 6 strain female mouse, and a pseudopregnant MCH strain female was injected. The mice were implanted into the uterus and implanted.

50% chimera rate among male chimeric individuals with brown coat appearing in black coat
Were determined to have the injected embryonic stem cells at an equivalent level in the germ line, and crossed with male mice of the C57BL / 6 strain. As a result, it was confirmed that a germline chimera was present in a chimeric individual created using two cloned embryonic stem cells.

(2) Obtaining a heterozygous mouse in which one copy of the FUT8 allele has been disrupted After breeding the germline chimera obtained in this section (1) until the age of 8 weeks, it became sexually mature C57BL /
The offspring were obtained by mating with 6 female individuals. Among the offspring, from a tail of an individual having a brown coat, a known method [Nucleic Acids Res.
earch), 3 , 2303, (1976)].
Southern blots were performed according to the method described.

The genomic DNA of the heterozygote was treated with the restriction enzyme PstI to obtain a wild-type FUT8 allele-specific fragment of about 7.5 Kb and homologous recombination-induced allele-specific fragment of about 11.
A fragment of 0 Kb was generated in a 1: 1 ratio (FIG. 2). As a result of Southern blot, this section (1)
It was confirmed that a heterozygote satisfying the above criteria was obtained from any of the chimeric individuals derived from any of the two embryonic stem cell clones whose contribution to the germ line was recognized (FIG. 3). This heterozygous mouse was a mouse in which one allele of FUT8 was disrupted.

(3) Obtaining homozygous mice in which both FUT8 alleles have been disrupted After breeding the heterozygous male and female individuals obtained in this section (2) until the age of 8 weeks,
They were crossed to obtain litters. Among the offspring, a known method [Nucleic Acids Research, 3 , 230] was used from the tail of an individual having a brown coat.
3, (1976)], and genomic DNA of each clone was prepared, followed by Southern blotting according to the method described in Example 3 (4).

The homozygous genomic DNA yields only a fragment of about 7.5 Kb specific to the wild-type FUT8 allele by treatment with the restriction enzyme PstI (FIG. 2). As a result of Southern blotting, it was confirmed that homozygotes meeting the above criteria were contained in the offspring (FIG. 3). This homozygous mouse was a double knockout mouse in which both FUT8 alleles were disrupted.

Embodiment 2. FIG. Expression analysis of transgenic mice lacking both α1,6-fucosyltransferase (FUT8) alleles Analysis of Gene Expression Amount in FUT8 Double Knockout Mice Small intestine, lung and brain were excised from FUT8 knockout homozygous mice and wild-type mice prepared in Example 1, Section 4, and subjected to molecular cloning, second edition [Molecula
r Cloning, A Laboratory Manual, 2 nd Ed. (1989)] and the like, and total RNA was prepared according to a known method. 2.2 mol / l using 20 μg of total RNA obtained from each organ
After performing 1.0% (w / v) agarose gel electrophoresis containing formamide, a known method [Proceedings of the National Academy of Sciences (Proc.
Natl. Acad. Sci. USA), 76 , 3683, (1979)] and a Zeta-probe membrane (B
RNA was transferred to IO-RAD).

On the other hand, the full length of human FUT8 cDNA [Journal of Biochemistry (J. Bioch.
em., 121 , 626 (1997)) was labeled with ³² P to prepare a probe. Molecular Cloning, 2nd edition [Molecular Cloning, A Laboratory Manual, 2 nd Ed. (1989
According to the known method described in [2], Northern hybridization was carried out by reacting the probe and the membrane prepared above at 55 ° C.

After hybridization, the membrane was immersed in 2 × SSC-0.1% (w / v) SDS,
Warmed for minutes. After repeating the above washing operation again, the washed film was exposed to an X-ray film at -80 ° C for 3 days and developed.
By this method, the expression of mouse FUT8 mRNA full length of about 3.5 Kb can be detected. As a result of this Northern blot, a single band of about 3.5 Kb was detected in all organs obtained from wild-type mice. On the other hand, in any organ obtained from the FUT8 double knockout mouse, no band corresponding to FUT8 mRNA could be detected (FIG. 4).
). From this, it was confirmed that FUT8 mRNA expression was lost in the FUT8 double knockout mouse.

2. Measurement of α1,6-fucosyltransferase activity of FUT8 double knockout mouse Brain and small intestine were excised from the FUT8 knockout heterozygous mouse, homozygous mouse and wild-type mouse prepared in Example 4, paragraph 4, and 4 times the amount Disintegrated in 0.25 mol / l sucrose-1.0 mol / l benzamidine-10 mmol / l Tris-HCl buffer (pH 7.4). After centrifugation at 900 × g for 10 minutes, the recovered supernatant was used as a crude enzyme. The enzymatic reaction was performed using 35 μl of a reaction solution (50 mmol / l MES-Na containing 30 μg to 90 μg of the crude enzyme).
OH buffer (pH 7.0), 0.3% Triton X-100, 0.285mmol / l GDP-fucose, 4.2μmol
/ l 4- (2-pyridylamino) butylamine-labeled (asparagine-linked) agalacto bia
ntennary sugar chain [Journal of Biological Chemistry (J. Biol. C
, 271 , 27810 (1996))] at 37 ° C for 2 hours. The reaction was stopped by heating at 100 ° C. for 1 minute. In addition, the reaction using the crude enzyme prepared from the FUT8 knockout homozygous mouse was measured by incubating for 12 hours. After the reaction was stopped by heating, 5000 x g
10 μl of the supernatant collected by centrifugation for 10 minutes was prepared as a sample. Then, L
A TSK-gel ODS-80TM column (4.6) attached to a C-VP HPLC system (manufactured by Shimadzu Corporation).
× 150mm, manufactured by Tosoh), and then injected with 0.15% 1-butanol-20mm
The fluorescence intensity (excitation wavelength: 320 nm, detection wavelength: 400 nm) of the eluted reaction product was measured using ol / l sodium acetate buffer (pH 4.0) developed at 55 ° C. at a flow rate of 1.0 ml / min. .

As a result of the enzyme activity measurement, no α1,6-fucosyltransferase activity was observed in the small intestine of the FUT8 knockout homozygous mouse, even when the reaction time of a wild-type mouse-derived organ was extended to 6 times. On the other hand, α1,6-fucosyltransferase activity corresponding to the expression level of the FUT8 gene was detected in the brains of FUT8 knockout heterozygous mice and wild-type mice (FIG. 5). From this, it was confirmed that α1,6-fucosyltransferase activity was lost in the FUT8 double knockout mouse.

FIG. 2 is a diagram showing a genomic region including an exon in which a translation initiation codon of a mouse FUT8 gene is located. Fig. 2 shows the structure of the targeting vector for disrupting mouse FUT8 gene and the method for determining Southern blot. Fig. 2 shows a genomic Southern blot of a mouse in which the FUT8 allele has been disrupted. Fig. 2 shows Northern blots using each organ of a mouse in which the FUT8 allele has been disrupted. Fig. 2 shows α1,6-fucosyltransferase activity in mice in which the FUT8 allele has been disrupted.

Claims (6)

The genome was modified such that the activity of an enzyme involved in sugar chain modification in which the 1-position of fucose was α-linked to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked complex type sugar chain was reduced or deleted. Mouse or its offspring. The mouse or the mouse according to claim 1, wherein a genomic gene of an enzyme involved in sugar chain modification in which position 1 of fucose is α-linked to position 6 of N-acetylglucosamine at the reducing end of N-glycoside-linked complex type sugar chain is knocked out. Its descendants. 2. All the alleles on the genome of an enzyme involved in sugar chain modification in which the position 1 of fucose is α-linked to the position 6 of N-acetylglucosamine at the reducing end of N-glycoside-linked complex type sugar chain were knocked out. Or the mouse or progeny thereof according to item 2. 4. The enzyme involved in sugar chain modification in which position 1 of fucose is α-linked to position 6 of N-acetylglucosamine at the reducing end of N-glycoside-linked complex type sugar chain is α1,6-fucosyltransferase. The mouse or progeny thereof according to any one of the above. The mouse or its progeny according to claim 4, wherein the α1,6-fucosyltransferase is a protein encoded by a DNA selected from the following (a) or (b):
(a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 2;
(b) a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having α1,6-fucosyltransferase activity. α1,6-fucosyltransferase has the following (a), (b) and (c)
The mouse or progeny thereof according to claim 4, which is a protein selected from the group consisting of:
(a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1;
(b) in the amino acid sequence represented by SEQ ID NO: 1, one or more amino acids are deleted,
A protein consisting of a substituted, inserted and / or added amino acid sequence and having α1,6-fucosyltransferase activity;
(c) a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having α1,6-fucosyltransferase activity;

Images