JP2001519145A - Transgenic animal having knock-in VEC receptor gene and use thereof - Google Patents

Abstract

  (57) [Summary] The present invention provides a non-human transgenic animal having cells that express a foreign DNA sequence encoding a functional vascular endothelial cell receptor domain but do not express a substantially homologous native DNA sequence. The invention also provides methods of using these transgenic animals to identify therapeutics and other reagents that affect angiogenesis. Further, the present invention provides a method using a transgenic animal having cells containing a donor gene as a model for testing various reagents.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a transgenic (gene transfer) having a “knock-in” gene.
Related to the development and use of animals. Such animals can be used to test substances such as small molecules, antibodies and other reagents used in human or veterinary medicine.

[0002]

[Prior art]

The knock-in method is a kind of gene replacement technology developed as a developmental research tool (Hanks et al., Science, vol. 269, Aug. 4, 1995).
, A variant of target gene inactivation (the "knockout" method). In a conventional transgenic approach, genes are introduced into an animal embryonic cell line at an early developmental stage. In the "knockout" method, an irrelevant DNA sequence is used for gene inactivation (
Mansour et al., Proc. Natl. Acad. Sci. USA 87: 768
8-7692 (1990); Sharaby et al., Nature, vol. 376, J
uly 6, 1995). In the knock-in method, the gene is not simply inactivated, as in the sometimes lethal knock-out method, and at the same time is replaced by another gene at that site by a known method such as homologous recombination. .
For example, in the knock-in method, cDNA derived from gene 1 is replaced with another gene (gene 2).
Gene 2 is inactivated while at the same time under the control of regulatory elements. One of the advantages of the knock-in method is that a gene can be introduced into the genome at a specific site. Also, knocking out a gene can be lethal to the animal, but replacing it with an appropriate homologous gene using the knock-in method allows the animal to survive.

The knock-in method has been used as a powerful tool for finding the function of specific genes in mammalian pathology. For example, as a result of a modification of the knock-in method, a part of the human MYH11 cDNA was homologously recombined into a Cbfb locus (lo
cus), the introduction of the human fusion oncogene CBFB-MYH11 obtained by knocking-in into the mouse deepened the understanding of the mechanism by which CBFB-MYH11 contributes to leukemia development (Castila et al., Cell, 87:
687-696, November 15, 1996).

[0004]

[Problems to be solved by the invention]

The invention described herein is the first disclosure of a knock-in technique used to replace a complete gene from one species with a corresponding homolog or chimeric construct of another species. In addition, this knock-in technique has been used to study the function of genes in mammals, and the invention described herein is the first knock-in technique used to develop an animal model for testing a therapeutic agent. It is disclosure of.

[0005] More specifically, the present invention also relates to the development of transgenic animals having a knock-in vascular endothelial cell-specific receptor gene. Heretofore, animal models for testing therapeutic agents specific for certain vascular endothelial cell-specific receptors have not been well established. According to the present invention, a test using a quantitative index for a therapeutic agent that inhibits angiogenesis can be routinely performed. In contrast, currently available in vivo model systems such as the SCID mouse model are difficult to use routinely. For example, the SCID mouse model requires cumbersome surgical procedures. In addition, this model often suffers from complications that are not easily controlled, such as the vascular system of the mouse causing unwanted angiogenesis in surgically implanted skin pieces.

The knock-in animal model of the present invention can be used to test the role of the vascular endothelial cell-specific receptor and the function of this receptor in pathologies such as tumorigenesis. Furthermore, the knock-in animal model of the present invention is useful for identifying therapeutic agents such as target molecules, antibodies, and other reagents that react only with certain types of molecules. One example of the knock-in animal model of the present invention is a model particularly useful for identifying an inhibitor of pathological angiogenesis. Such inhibitors are likely to be specific for the human vascular endothelial cell-specific receptor, but need to be tested in animal models that express the human receptor gene, such as the knock-in mice of the present invention.

[0007] In one aspect, the present invention relates to the development of a knock-in animal model useful for identifying an inhibitor specific to a vascular endothelial cell-specific receptor.
The background to the pathogenesis of angiogenesis and therapeutic approaches for inhibiting pathological angiogenesis are described.

[0008] Vascular endothelial cells (VECs) play an important role in angiogenesis during human development or in many conditions, such as tumorigenesis, ocular diseases, arthritis and arteriosclerosis. Neovascularization, the formation of new blood vessels during embryogenesis, involves multiple cells involved in endothelial cells, such as proliferation, migration, cell-cell interactions, cell-matrix interactions, morphological changes and tissue invasion. Controlled by the process. Angiogenesis, the formation of new blood vessels from pre-existing blood vessels, involves similar cellular processes, and the need for tissue infiltration of capillary endothelial cells of pre-existing blood vessels is also high. Both of these events, neovascularization and angiogenesis, are referred to herein as angiogenesis. Angiogenesis is important in normal physiological processes such as embryonic development, follicular proliferation, and wound healing, as well as tumor growth and neovascular glaucoma (J. Volkman and M. Kragsbrunn, M. Science 235: 442). −44
7 (1987)), non-neoplastic diseases related to abnormal neovascularization such as retinal neovascularization due to diabetes and macular degeneration due to aging, and also symptoms including chronic inflammatory diseases such as rheumatoid arthritis.

At present, there are few or no satisfactory treatments for many of these diseases. Therefore, vigorous research is under way to test the hypothesis that inhibiting the angiogenic components of these diseases is an effective treatment. Although many pharmacological agents with angiogenesis inhibiting properties have been identified, their clinical trials have not been sufficiently advanced. Furthermore, the mechanism of action of many known angiogenesis inhibitors is poorly understood. Therefore, there is a need for the development of a new class of angiogenesis inhibitors that act by inhibiting known specific angiogenesis-related mechanisms.

Angiogenesis involves mitogenesis, migration, adhesion (L. Kabada et al., J. Clin. In
vest. 98: 886-893 (1996); C. Brooks et al., Cel
179: 1157-1164 (1994)), and can be controlled by inhibiting various VEC functions such as invasion and maturation. One approach is to inhibit the action of major endothelial growth factors, especially vascular endothelial growth factor (VEGF).

[0011] The endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), acts as an angiogenesis-inducing agent by specifically promoting endothelial cell proliferation. V
EGF is a homodimeric glycoprotein consisting of two 23 kD subunits that are structurally similar to PDGF. By selective splicing of mRNA, VEG
There are several different monomeric isoforms for F. As the isoforms, two membrane-bound ones (VEGF ₂₀₆ and VEGF ₁₈₉ ) and a soluble two
There are species (VEGF ₁₆₅ and VEGF ₁₂₁ ). VEGF ₁₆₅ is the most abundant isoform in all human tissues except the placenta.

VEGF is expressed in embryonic tissue (Brier et al., Development (Camb.) 1).
14: 521 (1992)) and expressed in macrophages to proliferate epithelial keratinocytes during wound healing (Brown et al., J. Exp. Med., 1
76: 1375 (1992)) and is also associated with tissue edema associated with inflammation (Ferrara et al., Endocr. Rev. 13:18 (1992)). in situ
Hybridization studies have shown high VEGF expression in a number of human tumor lines, such as polymorphic glioblastomas, hemangioblastomas, central nervous system tumors and AIDS-related Kaposi's sarcoma (K. Plate et al., ( 1992) Natu
re 359: 845-848; Plate et al., (1993) Cancer
Res. 53: 5822-5827; Barkman et al. (1993) J. Mol. Cl
in. Invest. 91: 153-159; Nakamura et al., (1992) A
IDS Weekly, 13 (1)). High levels of VEGF have also been found in hypoxia-induced angiogenesis (D. Schweiki et al., (1992) Nature.
359: 843-845).

[0013] The biological response of VEGF was determined during embryonic development (B. Miralau et al., (1993) Ce
11 72: 835-846) and via high affinity VEGF receptors that are selectively expressed on endothelial cells during tumorigenesis. Recently, a number of receptor tyrosine kinases (RTKs) that are specifically expressed on endothelial cells have been cloned and characterized. While there are RTKs that are widely expressed on a variety of cell types, two families have been shown to be restricted primarily to endothelial cells and the primary hematopoietic system.

One family includes mouse FLK-1, its human homolog, K
VEGF receptors such as DR, FLT-1 and FLT-4 are included. FLK-
1 / KDR encodes a receptor for VEGF-A, VEGF-B and VEGF-C. FLT-1 and FLT-4 were VEGF-A and VEGF-A.
Each encodes a receptor for C.

[0015] Another family includes TIE-1 and TIE-2. TIE-
2 is also known as TEK. Angiopoietin-1, a ligand for TIE-2, has only recently been cloned (S. Davis et al., Cell 87).
: 1161-1169 (1996)). The ligand for TIE-1 has not yet been characterized.

[0016] VEGF receptors, particularly FLK-1 / KDR, have been considered to be strongly associated with angiogenesis associated with various human pathologies. In this context, a great deal of effort has been made to identify tumor angiogenesis inhibitors using the main target VEGF molecule and its receptor FLK-1 / KDR (KJ Kim et al., Nature).
362: 841-844 (1993); M. Strone et al., Cancer
Research 56: 3540-3545 (1996)).

[0017] VEGF receptors are usually class III receptor tyrosine kinases characterized by having usually seven immunoglobulin-like loops in the amino-terminal extracellular receptor ligand binding domain (Kaypainen et al., J.
. Exp. Med. 178: 2077-2088 (1993)). The other two regions include the transmembrane region and a carboxy-terminal intracellular catalytic domain that is interfered by the insertion of a variable length hydrophilic interkinase sequence, called the kinase insertion domain (Westermark et al., Prog. Growth). Factor
Res. 1 (4): 253-266 (1989); Terman et al., Oncogen.
e 6: 1677-1683 (1991)). VEGF binds to or activates its corresponding receptor, a tyrosine kinase expressed on the surface of endothelial cells, resulting in VEGF as an endothelial cell growth inducer.
Bring out the function of GF itself.

[0018] It is important that these two families of receptors play an important role in embryonic development.
Studies of knockout mice for each gene have shown this clearly. For example, F
LK-1 / KDR is important in endothelial cell differentiation and FLT-1 is important in organizing the major capillary plexus during early embryonic development. TIE-2 has also been shown to be important in remodeling vascular networks during angiogenesis in embryos,
IE-1 has been identified as a key molecule in the maturation of vascular networks (T.
N. Sato, Nature, 376: 70-74 (1995)). However, since the knockout of each gene causes embryonic lethality, the role of these receptors during late embryonic development and pathological conditions has not been studied. FLK-1 / KD in tumor angiogenesis
An important role for R is in the retrovirus-mediated dominant negative FLK-
It is clearly demonstrated that one gene transfer can prevent neovascularization in gliomas and consequently prevent tumor growth.

There are several reasons why KDR is a therapeutic target with very desirable properties. Of particular importance is that the KDR receptor is expressed almost exclusively in endothelial cells. KDR is also strongly up-regulated in activated (proliferating) endothelium, as opposed to resting endothelium. In addition, KDR is an accessible target because it is expressed on the surface of vascular cells. Therefore, drugs against the extracellular domain of KDR act very specifically and do not need to enter endothelial cells,
Also, there is no need to reach the inside beyond the vasculature to work effectively on the tissue,
Therefore, the effect can be obtained even at a low dose, which can be particularly useful. These advantages may also contribute to the favorable safety of the anti-KDR drug.

[0020] Due to these KDR properties, VEGF-KD is more at the level of KDR than at VEGF.
It has been shown to be advantageous to inhibit the R system. KDR is specifically localized on the surface of vascular cells. On the other hand, VEGF ligand is used in the interstitial space of tissue.
space) and exists in a wide range at a high concentration. Thus, VEGF ligands are often found in large quantities in association with heparan sulfate proteoglycans.

[0021] Inhibiting neovascularization by inhibiting the function of KDR can create novel therapeutics with potential success. This approach is also advantageous because it offers the potential to inhibit endothelial proliferation with high specificity, in contrast to the commonly used less specific treatments currently used. Also, in contrast to commonly used means of directly attacking tumor cells with drugs that have low specificity and are often cytotoxic, they are cytostatic, specific, and The use of non-toxic drugs to suppress the blood supply facilitates the control of malignant tumor growth. For example, specifically expressed on the surface of endothelial cells, as opposed to another target (eg, on tumor cells) that is deeply distributed at high concentrations in the interstitial space of the tissue It is advantageous to inhibit certain angiogenic receptors. More importantly, due to the availability of effective and non-toxic anti-angiogenic drugs, it is necessary to control the metastatic growth of tumors and various diseases such as, but not limited to, rheumatoid arthritis It is possible to provide a long-term or life-long treatment method.

Our studies have shown that neutralizing antibodies to FLK-1 and KDR are species-specific and therefore do not cross-react. One anti-KDR antibody is F
It has been shown that it has no effect on the binding between LK-1 and VEGF, and that certain anti-FLK-1 antibodies have no effect on the binding between KDR and VEGF. I have. Attempts to test such anti-KDR antibodies and other antagonists for anti-angiogenic effects in existing mouse tumor models would therefore be useless.

Since there were no satisfactory animal models until the present invention was made, it is difficult to test for human antigen-specific antibodies and other inhibitors of human angiogenesis, such as tumor angiogenesis. there were. Typically, when testing various anti-cancer therapeutics, a human tumor cell line is injected into immunodeficient nude mice, and the mice are treated with the anti-cancer therapeutic after a period of tumor growth. Anti-angiogenic approaches, such as inhibition of the receptor KDR, are unique because the target tissue is the host (eg, mouse) vasculature and not human tumor cells. For example, as noted above, many KDR-specific mouse monoclonal antibodies cannot function in such models.

[0024] Possible approaches to avoid this problem in testing human specific antagonists in a mouse tumor model with mouse vasculature include the following. (1) Search for tumor models in mammals other than mice that have the FLK-1 receptor showing high homology to KDR. (2) Chimeric human skin / SCI
D mouse xenograft model (PC Brooks et al., J. Clin. Invest.
. 96: 1815-1822 (1992)). However, these approaches are not enough. The advantages of using a mouse tumor model are clearly present. Numerous syngeneic and xenogeneic mouse models of tumor growth have been developed, and the relevance of FLK-1 in these models has been established (B. Milauer et al., Cancer Research 56: 1615-1620 (1996).
)). Substitution of other models (approach (1)) has the following main problems. (A) There is no guarantee that anti-KDR antibodies will cross-react with homologous receptors in other species, including primates. (B) It takes time to establish consistency and reproducibility and is probably difficult to achieve in tumor models other than mice. (C
) Satisfactory non-mouse tumor models are not common and are not readily available.

With respect to approach (2), the chimeric human skin / SCID mouse xenograft model is technically time consuming and difficult to quantify (PC Brooks et al., J.
Clin. Invest. 96: 1815-1822 (1995)). in addition,
There is additional expense regarding the cost of SCID mice, their maintenance, and the effort required to perform the experiments. Furthermore, limited information is available because the survival time of human skin grafts in mice is limited and processing time is limited. In addition, there is a problem that the vascularization of the skin graft due to the mouse vasculature increases as the skin graft grows, particularly when the graft has a tumor.

Thus, in order to test potential therapeutic molecules for human therapy in vivo, human receptor homologs (eg, FLK-1) can be substituted for native receptors (eg, FLK-1).
For example, a transgenic animal model expressing KDR) is needed. There is also a need for transgenic animal models that express homologous genes from other species in order to test potential veterinary therapeutic molecules in vivo. It is an object of the present invention to provide an animal model for testing the role of species-specific receptor tyrosine kinase in vivo. This strategy provides a unique approach to understanding the role of each endothelial cell-specific receptor tyrosine kinase during pathological angiogenesis and facilitates the identification of therapeutic target molecules.
It is another object of the present invention to provide a transgenic animal model for testing the efficacy and specificity of potential therapeutic reagents, particularly species-sensitive or species-specific reagents.

[0027]

[Means for Solving the Problems]

To achieve the above and other objects, the present invention provides a method of testing a substance for use in an animal, the method comprising expressing a foreign gene or a functional gene fragment from a different species. Administering the substance to a transgenic non-human animal having cells that do not express a substantially homologous native gene or functional gene fragment, and further evaluating the effect of the substance on the transgenic animal. In the present invention, the foreign gene and the native gene or gene fragment can encode a vascular endothelial cell receptor domain.

[0028] The present invention also provides transgenic non-human animals having cells that express foreign genes from different species but do not express substantially homologous native genes. More specifically, the present invention relates to transgenic non-human animals having cells that express a foreign DNA sequence encoding a functional extracellular vascular endothelial cell receptor domain, but do not express a substantially homologous native DNA sequence. provide.

[0029]

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides a transgenic animal having cells that express a foreign DNA sequence encoding a functional vascular endothelial cell receptor (VECr) domain. V
The ECr domain may be a VECr fragment, such as the extracellular portion of the receptor, or may be a whole VECr. The foreign DNA sequence is substantially homologous to the native DNA functional VECr domain sequence of the transgenic animal. The cells of the transgenic animal of the present invention are prepared using the homologous native DNA V
Does not express the ECr domain sequence.

The VECr foreign DNA sequence is composed of a fragment c
It can be DNA or complete cDNA. In addition, VECr foreign DNA
The sequence may encode a chimeric receptor whose DNA sequence may encode a different species of receptor. In one preferred embodiment, the extracellular portion of the receptor is obtained from a different species than the recipient animal.

The transgenic animal of the present invention can be produced using a knock-in method. In a preferred embodiment, the homologous recombination using a targeting vector containing a foreign DNA sequence results in
Insert the sequence while inactivating the native DNA sequence. As used herein, "substitution" and synonyms for "substitution" are defined as exogenous D at the homologous native DNA sequence site.
It refers to the insertion of the NA sequence and the concomitant inactivation of the native DNA sequence.
Inactivation of a native DNA sequence occurs when a foreign DNA sequence is inserted into a native gene and the exons of that gene are destroyed. The foreign DNA sequence is then placed under the control of the native promoter of the inactivated DNA sequence.

The transgenic animal of the present invention is preferably a member of a species different from the donor species of VECr encoded by foreign DNA. Preferably, both the transgenic animal and the donor are vertebrates, more preferably both are mammals. In one embodiment, the transgenic animal is a mouse,
Non-human mammals such as rats, pigs, goats, sheep or monkeys, and human donors. In a preferred embodiment, the transgenic animal is an animal normally used for medical or veterinary research, ie, an experimental animal. Experimental animals include mice, rats, rabbits, dogs, pigs, cows, horses, goats,
And sheep, but are not limited thereto.

In a more preferred embodiment of the invention, the donor DNA sequence is human and the transgenic animal is a mouse. In this preferred embodiment,
The human DNA sequence is preferably placed under the control of a mouse tissue-specific regulatory component, such as a mouse endothelial cell-specific promoter. In a preferred embodiment, the donor human VEC rDNA is constructed without a promoter. This promoterless VEC
The rDNA construct is directed using the vectors of the invention to a target in the mouse genome at a site downstream of the promoter for mouse VECr.

[0034] The receptor of the present invention may be any VEC receptor. Examples of VEC receptors include protein tyrosine kinase vascular endothelial growth factor (VEGF)
F) Receptors, namely KDR, FLK-1, FLT-1 and FLT-4
But are not limited to these. KDR is a human VEGF receptor and has a molecular weight of 180 kD. FLK-1 is a mouse homolog of KDR. FLT-1 is different from the KDR / FLK-1 receptor but has a related VEG
One type of F receptor. Both FLK-1 and KDR were VEGF-A
, VEGF-B and VEGF-C. FLT-1
And FLT-4 encode receptors for VEGF-A and VEGF-C, respectively. VEC receptors of the present invention also include TIE family receptor tyrosine kinases, including TIE-1 and TIE-2. TIE-2 encodes a receptor for angiopoietin-1 ligand.

In a preferred embodiment of the present invention, the mouse FLK-1 gene is a mouse FK-1 gene.
Under the control of the LK-1 promoter, it is replaced with KDR cDNA derived from a human donor. The mouse recipient obtained in this way contains native FLK-1 and KD
Both R receptors are expressed. Cross-bleeding these mouse recipients produces homozygous KDR / KDR mice.

In another embodiment of the present invention, the mouse FLK-1 gene is a mouse FLK
Under the control of the -1 promoter, is replaced by the chimeric KDR / FLK-1 cDNA.
In chimeras, the sequences encoding the extracellular and transmembrane domains of KDR are preferably fused to sequences encoding the intracellular domain of FLK-1, while the transmembrane domain is derived from KDR or FLK-1. These clones can be used to generate homozygous KDR / FLK-1 mice, and therefore, intracellular mouse F
The LK-1 domain is compatible with mouse cells.

The present invention also provides a transgenic animal having cells containing a donor gene obtained from a transgenic animal or a heterologous animal in which the substantially homologous native gene of the ancestor of the transgenic animal has been replaced, Here the cells no longer express the native gene. The transgenic animal is preferably a mouse, and the donor gene is preferably a human. The donor gene may be an animal gene or a synthetic version substantially similar to such a donor gene or a derivative thereof.

Usefulness The present invention relates to a method for testing a substance that specifically interacts with a protein expressed by a donor VEC rDNA sequence, which comprises administering the substance to the transgenic recipient of the present invention; A method comprising assessing the effect of the substance on For example, studies have shown that mouse VEGF binds and activates human KDR receptor, so that homozygous KDR / KDR mice of the present invention can be used for various small molecules, antibodies, and antibodies that affect angiogenesis. Useful as an animal model for testing other reagents. Such reagents may inhibit or enhance angiogenesis. Examples of small molecules that can affect angiogenesis include heterocyclic molecules, aromatic molecules, and oligopeptides, among others. Examples of antibodies that can affect angiogenesis include those well known in the art.

Further, the present invention relates to a method for identifying a substance capable of inhibiting abnormal angiogenesis, comprising administering the substance to the transgenic KDR animal of the present invention, wherein the substance inhibits abnormal angiogenesis. Providing a method that includes determining whether to do so. The present invention also provides a method for identifying a substance capable of inhibiting angiogenesis such as tumor angiogenesis, comprising administering the substance to the transgenic KDR animal of the present invention, and determining whether the substance inhibits angiogenesis. A method is provided that includes determining. The present invention also provides a method of identifying a substance capable of inhibiting tumor growth, comprising administering the substance to the transgenic animal of the present invention, and determining whether the substance inhibits tumor growth. Provide a way. The present invention also provides a method for identifying a substance capable of promoting wound healing, comprising administering the substance to the transgenic KDR animal of the present invention and determining whether the substance promotes wound healing. Provide methods including:

The present invention also provides the transgenic animal of the present invention used in a method for testing a substance used in human or veterinary medicine. Methods for testing a substance for use in humans include administering the substance to a transgenic non-human animal having cells containing the DNA sequence of a human donor. The donor (foreign) DNA encodes the particular gene of interest and replaces the substantially homologous native DNA sequence of the animal or animal ancestor such that the cell no longer expresses the native DNA sequence. The transgenic animal is then evaluated for the effect of the substance on the animal. In a preferred embodiment, the non-human transgenic animal is a mouse. Further, in another preferred embodiment, the donor DNA sequence is under the control of a tissue-specific regulatory component of the transgenic animal. Methods of testing a substance for use in veterinary medicine include cells containing the DNA sequence of a donor that has been replaced by a substantially homologous native DNA sequence of an animal or animal ancestor such that the cells no longer express the native DNA sequence. Administering the substance to a transgenic animal having the substance and evaluating the effect of the substance on the animal. In a preferred embodiment, the transgenic animal and the donor each belong to different species. In another preferred embodiment, the donor DNA sequence is under the control of a tissue-specific regulatory component of the transgenic animal.

DNA total RNA encoding the VEC receptor is prepared from endothelial receptor containing tissue by standard procedures. Total RNA is used for cDNA synthesis. Standard methods for isolating RNA and synthesizing cDNA are provided in standard manuals for molecular biology, such as: Sunbrook et al., "Molecular Cloning," Second Edition, Cold Sp.
ring Harbor Laboratory Press (1987), and Ausberg et al., (eds.) "Current Protocols in Molecular Biology", Green.
e Associates / Wiley Interscience, New
York (1990).

The complete gene or the cDNA of the receptor can be amplified by known methods.
Wear. For example, cDNA is used for amplification for polymerase chain reaction (PCR).
Used as plates (Saiki et al., Science,239, 487 (19
98) or Murris et al., U.S. Patent No. 4,683,195). For PCR amplification
The sequences of the oligonucleotide primers were the mouse and human VEGF
It is derived from the scepter sequence. Oligonucleotides are known in the art.
Synthesized by the method described above. A preferred method is that Carouser uses Science. 230 , 281-285 (1985).

Also, complete genes can be obtained by standard methods for isolating genomic clones from genomic phage libraries using standard hybridization techniques.

To isolate the entire protein coding region for the VEC receptor, an upstream PCR
The oligonucleotide primer is complementary to the sequence at the 5 'end and preferably comprises the ATG start codon and at least 5 to 10 nucleotides upstream of the start codon. Downstream PCR oligonucleotide primers are complementary to the sequence at the 3 'end of the desired DNA sequence. The desired DNA sequence is preferably VEG
It encodes the entire extracellular portion of the F receptor, optionally encodes all or a portion of the transmembrane region, and / or encodes all or a portion of the intracellular region including a stop codon. A mixture of upstream and downstream oligonucleotides is used for PCR amplification.
Conditions are optimized for each particular primer pair according to standard procedures. PC
The R product is analyzed by electrophoresis to confirm that the cDNA has the correct size corresponding to the sequence between each primer.

Alternatively, the coding region can be amplified in two or more overlapping fragments. Overlapping fragments are designed to contain restriction sites that allow for the assembly of a complete cDNA from this fragment.

The DNA encoding the VEC receptor of the present invention is inserted into a suitable target vector and inserted into a suitable recipient by homologous recombination. The DNA inserted into the recipient can encode the entire VEC receptor or a fragment of the VEC receptor.

A nucleic acid molecule encoding a VEC receptor or a portion thereof of the present invention can be inserted into a target vector using standard recombinant DNA techniques. Standard recombinant DNA techniques are described in Sambrook et al., "Molecular Cloning," 2nd Edition, Cold.
Spring Harbor Laboratory Press (1987)
And Ausberg et al. (Eds.) "Current Protocols in Molecular Biology", Gre.
en Publishing Associates / Wiley-Inter
science, New York (1990). A suitable source of cells containing nucleic acid molecules that express the VEC receptor includes VEC.

Suitable vectors for use in mammalian cells are known. Such vectors include well-known derivatives of SV-40, DNA sequences derived from adenoviruses, retroviruses, shuttle vectors derived from combinations of functional mammalian vectors as described above, functional plasmids and phage DNA. .

[0049]

【Example】

The following examples are described to facilitate understanding of the present invention, and the gist of the present invention is not limited by these examples. In the examples, detailed descriptions of conventional methods used for preparing vectors and plasmids, inserting genes encoding polypeptides into vectors and plasmids, or introducing plasmids into hosts are omitted. Such methods are well known to those skilled in the art and are described in Sunblock, E.C.
F. Frisch and T.W. Maniatis (1989) "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory
It has been described in a number of publications, including ory Press.

Knock-in of the KDR gene into mouse recipients In the homologous recombination step that occurs in mouse embryonic stem cells, the first exon of the FLK-1 gene is destroyed ("knock-out"), and at the same time, the KDR cDNA
("Knock-in"). The resulting heterozygous mice are expected to express both native FLK-1 and KDR receptors. Homozygous KDR / KDR mice are obtained by F1 intercross.

Experimental Design and Methods: Generation of Target Constructs : The following are the DNA components required for the targeting vector (FL
(K-1 genomic fragment and KDR cDNA). Also described is the preparation of a novel chimeric cDNA consisting of the full-length KDR construct and the extracellular portion of KDR fused to the intracellular portion of FLK-1.

FLK-1 genomic clone and cDNA clone: A 15 kb FLK-1 genomic clone is obtained. The 200 bp cDNA fragment containing the signal peptide sequence was prepared using the published FLK-1 sequence (W. Matthews et al., Proc.
tl. Acad. Sci. USA 88: 9026-9030 (1997)), and was obtained by PCR from a mouse lung cDNA library using primers. This fragment was randomly primed with 32P-dCTP and
The 9 / SV mouse genomic library (Stratagene) was used as a probe to screen. Phage DNA purified from clone # 8 was Sa
After digestion with II, the approximately 15 kb fragment was in both orientations.
ns) pBluescript SKII (+) vector (Stratagene)
To obtain the plasmid pmgFLK1.2. This clone was confirmed to encode mouse FLK-1 genomic DNA by restriction analysis (F. Sharaby et al., Nature 376: 62-66 (1995)).
Full-length FLK-1 cDNA is described in W.C. Matthews et al., Proc. Natl. Acad
. Sci. USA 88: 9026-9030 (1991).

KDR cDNA clone: The full-length KDR cDNA has the sequence described in the publication (BI.
Tarman, Oncogene 6: 1677-1683 (1991)), using primers complementary to RT-PCR. The template for RT-PCR includes human fetal kidney mRNA (Clontech) obtained from a spontaneously aborted human fetus.
) Was used. After obtaining two overlapping fragments encoding the 5 'and 3' regions of the cDNA, the expression vector pc was constructed using the unique BamHI site.
It was assembled in DNA3 (Invitrogen) to give the vector KB113.
The cDNA was completely sequenced on both strands.

Vectors for targeting constructs : neomycin (pPGK neo bpA) and thymidine kinase (TkpSL1190) (TN Sato et al., Nature 376).
: 70-74 (1995)).

Preparation of chimeric KDR / FLK-1 cDNA: FLK-1 and KDR cDNA share a unique BamHI site at the codon for methionine 806 in the KDR sequence. To amplify the sequence encoding the FLK-1 cytoplasmic domain, PCR primers are designed such that the 5 'primer is located just upstream of the BamHI site and the 3' primer is located just downstream of the stop codon. Furthermore, 3 '
Primers are designed to encode a NotI site. Full length FLK-1cD
NA acts as an amplification template. The PCR product was the vector pCR2.
1 (Invitrogen) and sequenced on both strands. FLK
-1 cDNA was digested with BamHI and NotI and the KDR expression vector KB11
3 (see above) and replaces the sequence encoding the KDR cytoplasmic domain. In the resulting chimeric cDNA, the first 20 of the cytoplasmic domain
Are from the human sequence. However, in this region, there is only one difference between the mouse and human proteins, the difference between glycine (KDR) and glutamic acid (FLK-1) at amino acid 794 of the KDR sequence.

Validation of the expression construct: The expression construct is tested for its ability to mediate the expression of a functional hybrid receptor molecule by transient transfection into COS7 cells. The full length KDR expression construct KB113 is used as a positive control. 48 hours after transfection, expression of KDR or KDR / FLK-1 is 125I
-Test in VEGF binding, fluorescence activated cell sorter (FACS), Western blot and receptor autophosphorylation assays.

In the 125I-VEGF binding, VEGF165 is iodinated with 125I. COS7 cells are seeded in a 24-well plate so that they are almost confluent. The following steps are all performed at 4 ° C. COS7 cells are treated with binding buffer (BB
= MCDB-131 / 15 mM HEPES / 0.1% gelatin / 1 μg / ml
After washing once with heparin), BB containing 2 ng / ml 125I-VEGF (
0.5 ml) for 2 hours. After incubation, the cells are
After washing three times with B, washing twice with PBS and dissolving in 1% Triton X-100, the radioactivity is counted.

In FACS, cells were removed with PBS containing 2 mM EDTA and 1% BSA (
Wash with cold Hanks balanced salt solution supplemented with HBSS-BSA) and then resuspend in the same buffer (100 μl) at a concentration of 105 cells per sample. Cells are incubated with 10 μg of the appropriate anti-KDR or control FLK-1 specific monoclonal antibody for 30 minutes. After washing, FITC (TAG
A 1:40 dilution of goat anti-mouse or anti-rat IgG conjugated with O) is added and incubated on ice for the last 30 minutes. The cells are then transferred to Coulter Ep
Analyze with ics Elite Cytometer. Data are for anti-FLK-1
Shown as a measure of the average fluorescence intensity of the anti-KDR monoclonal antibody binding to cells compared to a control measure of monoclonal antibody binding.

In Western blot analysis, transfected COS7 cells and control COS7 cells were 20 mM Tris-HCl pH 7.4, 1% N-octyl glycoside, 137 mM NaCl, 10% glycerol, 10 mM EDT.
A, 100 μg / ml Pefabloc (Boehringer Mannh
eim), dissolved in a buffer containing 100 μg / ml aprotinin and 100 μg / ml leupeptin. After centrifugation at low speed, the lysate is transferred to SDS-PAG.
Separate with E and transfer to nitrocellulose. KDR and chimeric KDR / FLK-1 receptor proteins are detected with an affinity purified polyclonal rabbit antibody developed at ImClone against the soluble KDR extracellular domain. Blot is 1
Incubate with 25I-labeled Protein A (Amersham) and detect by autoradiography.

In the receptor phosphorylation assay, control and transfected COS
Seven cells are starved for 24 hours in DMEM containing 0.5% CS, and then stimulated with 20 ng / ml VEGF for 10 minutes at room temperature. After ligand stimulation, cells are 1 mM
Washed with cold PBS containing sodium orthovanadate in 20 mM Tri
s-HCl pH 7.4, 1% N-octyl glycoside, 137 mM NaCl
Glycerol, 10% EDTA, 0.1 mM sodium orthovanadate, 10 mM NaF, 100 mM sodium pyrophosphate, 100 μg /
ml Pefabloc (Boehringer Mannheim), 100
Dissolve in a buffer containing μg / ml aprotinin and 100 μg / ml leupeptin. Spin down at 14,000 xg for 10 minutes, then immunoprecipitate the receptor from the clarified lysate using Protein A Sepharose beads conjugated to rabbit anti-KDR antibody. The beads are washed, mixed with SDS loading buffer and subjected to Western blot analysis. The phosphoprotein pattern of the stimulatory receptor is detected using an anti-phosphotyrosine monoclonal antibody (UBI) and developed by chemiluminescence (ECL; Amers ham).

All routine molecular biology procedures such as restriction digestion, ligation, PCR and Southern blotting are standard (J. Sunbrock, EF Frisch and T. Maniatis, Editor: "Molecular Cloning, Laboratory Manual"
Second Edition, Cold Spring Harbor Laboratory Pr
ess, Cold Spring Harbor, NY (1989)).

Construction of the Target Vector : A targeting vector targeting the FLK-1 locus and directing homologous recombination of two KDR receptor forms (full length KDR and chimeric KDR / FLK-1) is constructed as follows.

FIG. 1 outlines the strategy for disrupting the FLK-1 gene in mouse embryonic stem (ES) cells and expressing KDR or chimeric receptor under the control of FLK-1 regulatory components. The cDNA of the target construct in FIG.
Designated as R, this indicates either KDR or receptor chimera. Most of this strategy is based on the strategy used by Sharaby et al. And is used for targeted disruption of the FLK-1 gene (Nature 376: 62-66 (1995)).

The upstream FLK-1 genomic DNA fragment and KDR cDNA are assembled in a cloning vector designed for this study. The assembly vector consists of the pCRII backbone plasmid (Invitrogen) but its NsiI and Xb
The multiple cloning site (MCS) between the aI sites has been replaced by a synthetic MCS containing the required restriction sites in the following order (NotI, BamHI, NsiI, SmaI, KpnI, EcoRV and NotI). The correct structure of the assembled vector is confirmed by digestion with SmaI (since this enzyme is not present in the parental pCRII plasmid) and sequencing. The first coding exon (
1.8 kb genome F extending from the upstream BamHI site to the PstI site in FIG. 1)
The LK-1 fragment is cloned into the BamHI and NsiI sites of the assembly vector, while inactivating compatible PstI and NsiI sites. KDR cDNA and bovine growth hormone polyadenylation signal (pA)
The plasmid is excised from the KB113 expression plasmid with pnI and PvuI and cloned into the KpnI and EcoRV sites of the assembly vector (PvuII and EcoRV are compatible, they are blunt-cutting enzymes).

The NotI fragment and the KDR cDNA derived from the assembly vector containing the upstream FLK-1 genomic sequence were inserted into the unique NotI site of pPGK neobpA, and clones whose orientation of the inserted DNA matched those shown in FIG. select. Sal further downstream from the SmaI site downstream from the first coding exon
The 5.7 kb genomic FLK-1 DNA fragment extending to the I site was digested with H
It is cloned into the HindIII and SalI sites of pPGK neobpA by filling the indIII site with the Klenow fragment of DNA polymerase I. Finally, the blunted 2 kb HindIII fragment containing the thymidine kinase expression cassette is cloned into the blunted unique SacII site of pPGK neobpA.

The correct assembly of the final targeting vector is confirmed by PCR using primers located on adjacent DNA fragments and by restriction digestion.

Generation of mice with knock-in KDR gene : at least one allele of the FLK-1 gene has been inactivated and KDR or KDR / FLK-
Described below is a method of generating an ES cell line in which the expression of one chimeric mRNA is under the control of a native FLK-1 regulatory component.

The target vector was prepared by using an ECM600 electroporator (Gentronics) in HEPES-buffered saline at 160 V, a capacitance of 50 μF, and a resistance of 360 Ω under the conditions of 129 / svES (A. Nazy et al., Proc.
. Acad. Sci. USA 90: 8424-8428 (1993)). After electroporation, 2 × 104 cells are cultured in 100 mm dishes containing feeder STO fibroblasts (SL Mansour et al., Proc. Natl. A.
cad. Sci. ScL USA 87: 7688-7792 (1990)). 48 hours after electroporation, cells are selected using ganciclovir and G418, and each colony is separated.

[0069] Genomic DNA from the double selection ES clone is prepared and tested for homologous recombination by Southern blot. Genomic DNA is digested with NcoI or XhoI. Probes are generated from the FLK-1PstI-XhoI fragment downstream of the target locus (see FIG. 1) and are labeled with 32P. This probe was used for the 6
. A 5 kb NcoI fragment (F. Sharaby et al., Nature 376:
62-66 (1995)), and the detection of rather large fragments of the target locus obtained by insertion of KDR and neocDNA. Similarly, this probe detects an approximately 3.8 kb XhoI fragment generated by insertion of a new XhoI site just downstream of the KDR cDNA.

Subsequently, using the target ES cells, the chimeric KD was obtained under the control of the FLK-1 promoter.
Mice expressing R / FLK-1 or full length KDR receptor are obtained. First, a heterozygous germline chimera (KDR / FLK
-1) / FLK-1 or full length KDR / FLK-1 mice can be generated. Expression of KDR on the endothelium of heterozygous mice is confirmed by immunocytochemistry using KDR-specific polyclonal and monoclonal antibodies, and RT-PCR using KDR-specific primers. The mice were then cross-bred and homozygous full length KDR / KDR mice or homozygous chimeras (KDR / F
LK-1) / (KDR / FLK-1) mice are generated.

These mice can be cross-propagated with immunodeficient mouse lines such as RAG − / − and their progeny can be used as recipients for transplantation of various mouse and human tumor cell lines. The efficacy of administering a monoclonal antibody that inhibits KDR-VEGF binding, or administering another KDR-directing agent, can then be determined.

Transgenic Animal Models with Knock-In VEC Receptors and Their Use The following assays can be used to identify target molecules for therapeutic intervention. Also, the angiogenesis model described below can be used to test the resulting therapeutic reagents for their efficacy and specificity.

A. Tumor angiogenesis The mouse having the above knock-in VECr gene is crossed with a RAG-1 (-/-) mouse, and the obtained progeny is used for transplantation of a tumor cell line. Various human tumor cell lines are injected into the immunocompromised knock-in mice and the effects of therapeutic antibodies, target molecules and other human species-specific reagents are evaluated.

B. Ocular Neovascularization Locally inducible expression of each knock-in form of the receptor tyrosine kinase was tested in mice with a knock-in VECr gene during ocular neovascularization induced by various angiogenic factors such as VEGF and FGF. I do. These models are useful in studying ocular conditions such as retinopathy.

C. Acute and chronic inflammation models Mice with the above knock-in VECr gene are used to study the effects of therapeutic agents on various induced inflammations. These knock-in mice in which inflammation has been induced are particularly useful for studying the treatment of various acute and / or chronic inflammatory conditions such as rheumatoid arthritis.

D. Psoriasis model Psoriatic skin is characterized by microvascular hyperpermeability and angioproliferation. The proliferative epidermis of psoriatic skin expresses very large amounts of vascular endothelial cell growth factor. Thus, mice with the above-described knock-in VECr gene are useful in studying therapeutics for psoriasis often characterized by increased vascular endothelial cell growth factor.

E. Bullous disease Vascular endothelial cell growth factor is strongly expressed by epidermal keratinocytes during bullous diseases such as erythema multiforme and pemphigus pemphigoid. These conditions are characterized by increased microvascular permeability and angiogenesis. The development of erythema due to vascular hyperpermeability is also a common feature after overexposure to the sun. Mice having the above-mentioned knock-in VECr gene are useful for testing various therapeutic compounds that are effective against these various disease states.

F. Wound healing The role of angiogenesis in wound healing is well known. In particular, increased VEGF expression in wound healing has already been reported. Since the expression receptor is involved in wound healing, mice having the above-described knock-in VECr gene are useful for testing the effects of agonists and antagonists on the expression receptor. Such effects will enhance the understanding of the wound healing process and will also allow for therapeutic intervention in this process.

G. Arteriovenous Malformation Arteriovenous malformation (AVM) is a congenital disorder composed of abnormal vasculature, which is clinically important because it lacks capillary components and tends to cause spontaneous bleeding. TIE, an endothelial cell-specific receptor tyrosine kinase, has been shown to increase in AVMs and the surrounding cerebral vasculature. In addition, VEGF mRNA up-regulation was observed in brain parenchymal cells adjacent to AVM, and VEGF protein was detected in this tissue as well as AVM endothelium. In contrast, little or no TIE or VEGF is expressed in normal brain. V in AVM
Significant up-regulation of EGF and TIE implies ongoing angiogenesis that contributes to AVM hypoproliferation and maintenance. Therefore, mice having the above-mentioned knock-in VECr gene can be used to study the effects of therapeutic agents for these congenital disorders.

[Brief description of the drawings]

FIG. 1 shows a targeting strategy for knocking in KDR cDNA into the FLK-1 locus. This overview is a modification of the FLK-1 knockout targeting strategy of Sharabee et al. (Nature, vol. 376, July 6, 1995). The black rectangle indicates the NotI fragment containing the upstream FLK-1 genomic region. K
The DR cDNA and polyadenylation signal are assembled in an intermediate vector. Shown is the approximate length of the original genomic FLK-1 DNA and restriction fragments relevant to Southern blotting. Restriction enzymes: B, BamHI; H, HindIII; N,
NcoI; Nt, NotI; P, PstI; S, SmaI; SI, SalI; X
, XhoI.

FIG. 2 shows a targeting strategy for knocking in a KDR / FLK1-1 chimeric cDNA into the FLK-1 locus. This overview is based on the FLAK-1 knockout targeting strategy of Sharaby et al. (Nature, vol. 376, July 6, 199).
This is a modification of 5). KDR / FLK-1 chimeric cDN containing transmembrane region (TM)
A and the polyadenylation signal are assembled in an intermediate vector. Shown is the approximate length of the original genomic FLK-1 DNA and restriction fragments relevant to Southern blotting. Restriction enzymes: B, BamHI; H, HindIII; N, NcoI;
Nt, NotI; P, PstI; S, SmaI; SI, SalI; X, XhoI
.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/15 G01N 33/50 Z 33/50 C12N 15/00 A United States 10034 New York, New York, West 215 Street 583, Apartment 2F (72) Inventor Boren, Peter United States 10028 New York, New York, East 80 Street 178 (72) Inventor Sato, Thomas United States 75019 Texas Drive, Bradford Drive, Kappel, Oregon 1306

Claims (21)

[Claims] 1. Exogenous D encoding a functional vascular endothelial cell receptor domain
A non-human transgenic animal having cells that express the NA sequence but do not express the substantially homologous native DNA sequence. 2. The transgenic animal according to claim 1, wherein the transgenic animal is an experimental animal. 3. The transgenic animal according to claim 2, wherein the experimental animal is a mouse. 4. The transgenic animal according to claim 1, wherein the foreign vascular endothelial cell receptor DNA sequence is chimeric. 5. The transgenic animal according to claim 4, wherein the chimeric DNA sequence is a FLK-1 / KDR gene. 6. The transgenic animal according to claim 1, wherein the foreign DNA sequence is mammalian. 7. The transgenic mouse according to claim 3, wherein the foreign DNA sequence is human. 8. The transgenic mouse of claim 7, wherein the foreign DNA sequence that is human is under the control of a mouse tissue-specific regulatory component. 9. The transgenic mouse according to claim 8, wherein the human foreign DNA sequence is under the control of a mouse endothelial cell-specific promoter. 10. The transgenic mouse according to claim 9, wherein the promoter is FLK-1 or TIE-2 promoter. 11. The method according to claim 1, wherein the promoter is FLK-1 promoter.
The transgenic mouse according to 0. 12. The transgenic mouse according to claim 7, wherein the native DNA sequence is a FLK-1 gene. 13. The human foreign DNA sequence is a KDR gene.
The transgenic mouse according to claim 12. 14. The transgenic mouse according to claim 7, wherein the foreign DNA sequence is an extracellular human KDR gene fragment. 15. A non-human transgenic animal having cells that express foreign genes from different species but do not express substantially homologous native genes. 16. A method for testing a substance that interacts with a protein expressed by a foreign DNA sequence according to claim 1, wherein the substance is administered to the transgenic animal according to claim 1, and the animal. And estimating the effect of said substance in the method. 17. A method for identifying a substance capable of inhibiting angiogenesis, comprising administering the substance to the transgenic animal according to claim 1 and determining whether the substance inhibits angiogenesis. A method that includes doing. 18. A method for identifying a substance capable of inhibiting tumor growth, comprising administering the substance to the transgenic animal according to claim 1, and determining whether the substance inhibits tumor growth. A method that includes doing. 19. A method for testing a substance for use in an animal, the method comprising the step of converting the substance expressing a foreign gene or a functional gene fragment derived from a different species, but substantially homologous to a native gene or a functional gene fragment. A method comprising administering to a non-human transgenic animal having non-expressing cells, and evaluating the effect of said substance in said animal. 20. The method of claim 19, wherein the non-human transgenic animal is a mouse. 21. The method of claim 19, wherein the donor DNA sequence is under the control of a tissue-specific regulatory component of the transgenic animal.