WO2011040417A1 - イノシトール-1,4,5-トリスリン酸受容体結合タンパク質およびそれを含むトランスジェニック非ヒト哺乳動物 - Google Patents
イノシトール-1,4,5-トリスリン酸受容体結合タンパク質およびそれを含むトランスジェニック非ヒト哺乳動物 Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K2217/20—Animal model comprising regulated expression system
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- A—HUMAN NECESSITIES
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- A01K2227/00—Animals characterised by species
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- A01K2227/105—Murine
Definitions
- the present invention relates to an inositol-1,4,5-trisphosphate receptor (IP3R) binding protein and a transgenic non-human mammal containing the same.
- IP3R inositol-1,4,5-trisphosphate receptor
- KRAP gene encoding KRAP protein (Ki-ras-induced actin-interacting protein) is expressed by the present inventor as one of the genes whose expression level is up-regulated by activated Ki-ras. It was identified from the cDNA library of the strain HCT116 (Non-patent Document 1). The structure of KRAP gene is conserved from fish species to mammalian species beyond animal species, but its expression pattern and molecular function have not yet been elucidated.
- the present inventors made a specific polyclonal antibody against KRAP and examined the histological expression in mouse tissue.
- the KRAP protein showed ubiquitous expression in mouse physiological tissue. It was reported that the expression level was high in pancreas, liver and brown adipose tissue, and coexisted with filamentous actin along the apex of the membrane in pancreas and liver tissue (Non-patent Document 2).
- Subfraction studies of KRAP protein revealed that KRAP protein is a cytoplasmic protein, most of which is related to the cytoskeleton.
- the microarray gene expression profile with suppressed KRAP expression in the colon cancer cell line HCT116 shows that several types of receptors and signal molecules that are often deregulated in cancer cells are not knocked out in KRAP knockdown cells. It was revealed that expression was changed compared to down cells.
- the KRAP-deficient mouse prepared by the present inventor exhibits significant systemic energy metabolism abnormalities, and KRAP protein increases energy consumption, increases insulin sensitivity, prevents and eliminates obesity, and / or levels of hormone in blood It has been clarified that it has an effect of inducing abnormality (Patent Document 1, Non-Patent Document 3). Therefore, a drug targeting KRAP or a KRAP-related pathway may be a prophylactic and therapeutic drug for metabolic diseases such as obesity or diabetes. However, details of the molecular function of KRAP protein were unknown.
- the present inventor made a KRAP overexpressing mouse and identified it from the mouse liver tissue by co-purifying a protein that interacts with the KRAP protein.
- This binding protein is an inositol-1,4,5-trisphosphate (IP3) receptor binding protein, and the KRAP protein that interacts with the IP3 receptor (IP3R) is important for regulating the function of IP3R.
- IP3R IP3 receptor
- the object of the present invention is to provide an inositol-1,4,5-trisphosphate receptor (IP3R) binding protein, preferably an inositol-1,4,5-trisphosphate receptor (IP3R) binding KRAP protein. .
- IP3R inositol-1,4,5-trisphosphate receptor
- the amino acid sequence constituting the protein to which IP3R binds is a full length or an IP3R binding site-containing region containing an amino acid site to which IP3R binds, that is, an amino acid partial sequence region. It aims to provide an IP3R binding protein.
- the IP3R binding site-containing region is an amino acid sequence region consisting of 19 amino acids from the 200th to the 218th of mouse KRAP protein, or derived from an animal species other than mouse KRAP protein.
- an object is to provide an IP3R binding protein consisting of an amino acid sequence region corresponding to the amino acid sequence region of the mouse KRAP protein.
- the inositol-1,4,5-trisphosphate receptor binding protein or the structural mimetic compound synthesized based on the amino acid primary structure or higher order structure information of the IP3R binding site-containing region It is an object of the present invention to provide an inositol-1,4,5-trisphosphate receptor binding protein characterized by
- an intracellular calcium ion modulator characterized by comprising the IP3R binding protein or a binding site-containing region thereof as a main component, such as an IP3R function regulator, an IP3R activity inhibitor or an IP3R activity
- a main component such as an IP3R function regulator, an IP3R activity inhibitor or an IP3R activity
- the KRAP-IP3R protein interaction by preparing a KRAP-IP3R protein complex or IP3R binding site-containing region between KRAP protein and IP3R or detecting its binding ability It is an object of the present invention to provide an assay method for screening a substance that inhibits.
- IP3R binding protein or a related term refers to the binding between an IP3R protein and a protein such as KRAP protein.
- KRAP-IP3R protein complex it should be construed to include the KRAP-IP3R protein complex in which the base sequence constituting the IP3R protein and the protein such as KRAP protein and the amino acid sequence are combined.
- binding refers to binding in the form of association between base sequences or amino acid sequences by intermolecular action in addition to proteins. It means that you are doing.
- the present invention provides an inositol-1,4,5-trisphosphate receptor (IP3R) binding protein, preferably an inositol-1,4,5-trisphosphate receptor (IP3R) binding protein.
- IP3R inositol-1,4,5-trisphosphate receptor
- the amino acid sequence constituting the protein to which IP3R binds is a full length or an IP3R binding site-containing region including an amino acid site to which IP3R binds, that is, an amino acid partial sequence region.
- An IP3R binding protein comprising:
- the IP3R binding site-containing region is an amino acid sequence region consisting of 19 amino acids from the 200th to the 218th of mouse KRAP protein, or derived from an animal species other than mouse KRAP protein.
- an IP3R binding protein comprising an amino acid sequence region corresponding to the amino acid sequence region of the mouse KRAP protein is provided.
- the inositol-1,4,5-trisphosphate receptor binding protein or the structural mimetic compound synthesized based on the amino acid primary structure or higher order structure information of the IP3R binding site-containing region An IP3R binding protein consisting of
- an intracellular calcium ion modulator characterized by comprising the IP3R binding protein or a binding site-containing region thereof as a main component, such as an IP3R function regulator, an IP3R activity inhibitor or an IP3R activity Providing an agent.
- the KRAP-IP3R protein interaction by preparing a KRAP-IP3R protein complex or IP3R binding site-containing region between KRAP protein and IP3R or detecting its binding ability
- An assay method is provided that can screen for substances that inhibit the expression.
- the present invention provides a transgenic non-human mammal containing an IP3R binding protein that overexpresses KRAP protein. Furthermore, this invention provides the vector containing IP3R binding protein as another aspect.
- heparin heparin, xestospongin, and 2-aminoethoxydiphenyl borate (2-APB) are known as inhibitors of IP3R activity. Heparin also causes uncoupling of G protein signals and activation of ryanodine receptors, which is problematic in terms of specificity. Moreover, since there is no membrane permeability, injection into cells is necessary. Zestspondin is expensive because it is an alkaloid derived from sponges. Moreover, although the inhibitory effect of Ca 2+ signaling has been reported, the mechanism of action is not clear.
- IP3R-binding protein of the present invention is known in the art because it has the effect of regulating the function of IP3R by intermolecular action of proteins such as KRAP protein on IP3R. Therefore, it can be expected to be a new IP3R activity inhibitor having a different action and effect from the above IP3R activity inhibitors.
- the present invention relates to an IP3R function regulator, further an intracellular calcium ion concentration regulator, based on a substance that mimics the amino acid primary structure or higher-order conformation of the amino acid sequence of a protein such as KRAP protein and its IP3R binding site-containing region. Can be used for development.
- FIG. 1 is a diagram showing the results of PCR using ex ⁇ Taq enzyme using genomic DNA prepared from the tail of an established transgenic mouse as a template.
- FIG. 2 shows the results of Western blotting of a protein solution prepared from each tissue of a transgenic mouse and detection with an anti-HA antibody.
- FIG. 3 shows a diagram comparing the overexpression level of KRAP with that of the wild type in the liver.
- FIG. 4 is a view showing the results of examining the localization of the overexpressed KRAP protein in the liver by immunohistochemical staining using an anti-HA antibody.
- FIG. 1 is a diagram showing the results of PCR using ex ⁇ Taq enzyme using genomic DNA prepared from the tail of an established transgenic mouse as a template.
- FIG. 2 shows the results of Western blotting of a protein solution prepared from each tissue of a transgenic mouse and detection with an anti-HA antibody.
- FIG. 3 shows a diagram comparing the overexpression level of KRAP with that of the wild type in the liver
- FIG. 5 is a diagram showing an image of silver-stained HA-matrix binding fractions prepared from the livers of wild-type (WT) mice and transgenic (TG) mice by SDS-PAGE.
- FIG. 6 is a diagram showing the analysis result of the co-purified protein band using a mass spectrometer.
- FIG. 7 shows a Western blot of the co-purified product. It is a figure which shows the result by the Western method about the immunoprecipitation product by the anti- KRAP antibody from the protein extract of a tissue (a liver, a kidney, brown adipose tissue (BAT), pancreas).
- FIG. 10 is a diagram showing the results of immunocytostaining cultured cells (HEK293 cells) in order to observe protein co-localization.
- FIG. 11 is a diagram showing a representative example of calcium indicator fluorescence signal changes with time in stimulation with 1 ⁇ M, 3 ⁇ M, and 10 ⁇ M ATP final concentrations in KRAP gene expression-suppressed cells and non-suppressed cells.
- the upper side shows scramble siRNA-treated cells
- the lower side shows KRAP-suppressed cells.
- FIG. 12 is a diagram showing a comparison of maximum fluorescence values after ATP stimulation at ATP final concentrations of 1 ⁇ M, 3 ⁇ M, and 10 ⁇ M.
- the left side shows scramble siRNA-treated cells
- the right side shows KRAP-suppressed cells.
- FIG. 13 is a diagram showing a maximum fluorescence increase degree (Mean peak amplitude) in which the maximum fluorescence value is represented by the ratio with 1 being before ATP stimulation.
- the left side shows scramble siRNA-treated cells
- the right side shows KRAP-suppressed cells.
- FIG. 15 is a Western blot diagram showing suppression of KRAP expression in HEK293 cells.
- FIG. 16 is a schematic diagram of mouse KRAP protein.
- FIG. 17 is an electrophoretogram showing the total length of KRAP and the ability to bind each deletion mutant to IP3R1 in HEK293 cells.
- FIG. 18 is an electrophoretogram showing the total length of KRAP and the ability to bind each deletion mutant to IP3R1 in HCT116 cells.
- FIG. 19a shows the amino acid sequences of mouse KRAP and human KRAP for a 19 amino acid long region that is a KRAP protein region important for binding to IP3R identified in Example 5.
- FIG. 19b is an electrophoretogram showing the ability of a point mutant to bind to IP3R1.
- the present invention relates to regulation of intracellular calcium ion concentration such as an inositol-1,4,5-trisphosphate receptor (IP3R) binding protein, an IP3R binding protein, such as an IP3R function regulator, an IP3R activity inhibitor, and an IP3R activator. And a transgenic non-human mammal comprising an IP3R binding protein.
- IP3R inositol-1,4,5-trisphosphate receptor
- IP3R binding protein according to the present invention is widely present in various tissues and cells of non-human mammals in a form in which an IP3R protein and a protein such as KRAP protein are bound by intermolecular action.
- IP3 inositol-1,4,5-trisphosphate
- IP3R IP3 receptor
- IP3 also plays an important role in the control of various cell functions such as secretion, fertilization, muscle contraction, nerve signaling and cell growth (Berridge, M., et al., Nature, 361, 315, 1993; Claphem, D. E., Cell, 80, 259, 1995).
- This IP3 has the action of binding to IP3R present on the membrane such as the endoplasmic reticulum, which is a calcium ion reservoir, as a ligand, activating IP3R, causing calcium ion release, and increasing the intracellular calcium ion concentration. is doing.
- IP3R present on the membrane
- the endoplasmic reticulum which is a calcium ion reservoir
- activating IP3R causing calcium ion release, and increasing the intracellular calcium ion concentration.
- Calcium ions play an important function as intracellular signal transmitters, and the concentration of intracellular calcium ions is usually kept at a very low level of about 10,000 times extracellular. Changes are involved in various cellular responses such as cell division, cell death, fertilization, and development.
- IP3 receptor is widely distributed in various tissues and cells such as brain, heart, liver, kidney, pancreas and thymus of mammals such as humans and mice, and excitement of nerves and muscles. It exists not only in sex cells but also in non-excitable cells and is involved in a wide variety of life phenomena. IP3R is a tetrameric intracellular IP3-inducible Ca 2+ release channel (IP3-gated Ca ++ Release Channel) with three functionally different regions: an IP3 binding domain near the N-terminus and 6 near the C-terminus. It consists of a channel-forming domain having a transmembrane region and a control region between these two regions.
- IP3-gated Ca ++ Release Channel IP3-gated Ca ++ Release Channel
- IP3R has been examined in detail about the localization of IP3R in the brain so far, and it has been found that types 1, 2, and 3 exist in the brain. Among them, type 1 IP3R (IP3R1) is particularly abundant in Purkinje cells of the cerebellum and is known to show strong staining.
- IP3R binding protein is one type of binding protein in which a protein such as KRAP protein is bound to IP3R by intermolecular action.
- IP3R-binding KRAP protein can be obtained by co-purification from various tissues (eg, liver tissue) and cells of transgenic non-human mammals such as transgenic mice that overexpress the KRAP protein newly produced by the present inventors. Obtainable.
- the nucleotide sequence of human KRAP is SEQ ID NO: 1
- the amino acid sequence of human KRAP is SEQ ID NO: 2
- the nucleotide sequence of mouse KRAP is SEQ ID NO: 3
- the amino acid sequence of mouse KRAP is SEQ ID NO: 4
- the nucleotide sequence of human IP3R is SEQ ID NO: 5.
- the amino acid sequence of human IP3R is represented by SEQ ID NO: 6
- the nucleotide sequence of mouse IP3R is represented by SEQ ID NO: 7
- the amino acid sequence of mouse IP3R is represented by SEQ ID NO: 8.
- mouse KRAP protein (SEQ ID NO: 3) that binds to IP3R used in the present invention has a length of 1,252 amino acids, and the C-terminal region is presumed to have a coiled-coil structure. Therefore, in this invention, the IP3R-binding KRAP protein has not only the full length thereof, but also the IP3R binding site-containing partial region containing an IP3R binding site shorter than the full length has an activity like the full-length amino acid sequence of the IP3R-binding KRAP protein. In this case, it can be understood that the IP3R binding site-containing partial region is also included in the scope of the present invention.
- the KRAP protein region important for binding to IP3R can be determined by examining the binding ability of KRAP-deficient mutants to IP3R.
- KRAP full-length or deletion mutant is expressed together with IP3R in HEK293 cells, the protein extract is immunoprecipitated with anti-HA antibody, and whether IP3R co-precipitates is examined. The binding ability of can be examined.
- the 19 amino acid length region of amino acid numbers 200 to 218 of mouse KRAP protein is considered to be an important region for binding to IP3R. It can be understood that a region having an amino acid length including the region is also included in the scope of the present invention.
- the 19 amino acid long region having the amino acid number from 200 to 218 is considered to be an important region for binding to IP3R.
- the region corresponding to the IP3R binding region of mouse KRAP protein is considered to be an important region for IP3R binding.
- IP3R-KRAP protein complex which is the IP3R-binding KRAP protein according to the present invention or a structural mimetic or compound synthesized based on the amino acid primary structure or higher-order structure information of the IP3R binding site-containing region is IP3R binding. Since the KRAP protein or its IP3R binding site-containing region has substantially the same functions and effects, it is understood that these structural mimetics and compounds are naturally included in the scope of the present invention. Should.
- IP3R binding KRAP protein obtained by this invention and the substance having the amino acid partial sequence region of the IP3R binding KRAP protein are developed as intracellular calcium ion concentration regulators such as IP3R function regulators, IP3R inhibitors or IP3R activators. Can be used.
- an assay for screening a substance that inhibits the interaction between KRAP-IP3R protein by preparing a KRAP-IP3R protein complex between KRAP protein and IP3R or detecting its binding ability can also be used as a method.
- non-human mammals that can be produced in the present invention
- all mammalian species can be technically targeted.
- monkeys, cows, pigs, dogs, cats, rabbits, guinea pigs, rats , Hamsters, mice, etc. but rodents such as guinea pigs, rats, hamsters, mice, etc. are preferred from the viewpoint of ease of production, breeding and use, and many inbred strains have been created.
- Mice that are equipped with techniques such as culturing fertilized eggs and in vitro fertilization are the best.
- a method for producing a transgenic non-human mammal according to the present invention will be described.
- a mouse will be described as an example of a non-human mammal for the sake of brevity.
- the present invention is not limited to mice and can be applied to other non-human mammals as well unless otherwise specified.
- the transgenic non-human mammal of the present invention can be produced by introducing a target gene into a non-human mammal according to a known method commonly used in the art (for example, Proc. Natl. Acad). (See Sci. USA 77; 7380-7384, 1980).
- cDNA As the KRAP gene which is a transgene.
- This KRAP cDNA is synthesized from oligonucleotides based on a known mouse cDNA sequence or an arbitrary nucleotide sequence of a human cDNA sequence and used as a probe to screen a cDNA library, normal cells, cultured cells, etc. From the registered nucleotide sequence of KRAP cDNA ⁇ , oligonucleotides that hybridize to both ends of the target cDNA fragment are synthesized as forward and reverse primers. It can also be prepared by RT-PCR method.
- a promoter sequence or an enhancer sequence for controlling the expression can be linked to the transgene.
- the promoter sequence and enhancer sequence are not particularly limited, and a promoter region or enhancer region of a gene highly expressed in various organs of a transgenic animal can be appropriately selected and used.
- the gene obtained by the transgene cloning can be used to create an expression construct by introducing it into an expression vector containing an appropriate promoter.
- the transgene may be a recombinant gene linked downstream of an appropriate mammalian promoter, and a poly A signal may be linked downstream of the gene.
- the recombinant gene may be linked with a terminator necessary for expression of the gene encoding the peptide, and can also be used as a sequence (so-called poly A) that terminates transcription of the target mRNA. .
- the type of the promoter and poly A signal is not particularly limited, and examples thereof include promoters derived from viruses such as cytomegalovirus, JC virus, and breast cancer virus, humans, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice, and the like. Promoters derived from various mammals can be used.
- the promoter for example, a structure in which a cytomegalovirus enhancer and a chicken- ⁇ -actin promoter are linked and a pCAGGS vector containing a poly A signal site of a rabbit ⁇ -globin gene, a so-called CAG promoter, Is preferable because it can be overexpressed almost systemically.
- the terminator for example, a simian virus SV40 terminator can be used as the terminator.
- the expression construct thus prepared is injected into Escherichia coli, amplified by culturing the Escherichia coli, purified, and then confirmed to have a correct base sequence with a sequencer.
- the expression construct produced as described above is usually injected into one of the pronuclei before fusion of the male and female nuclei of fertilized eggs of non-human mammals such as mice using a microinjection device.
- the target gene can be introduced.
- the prepared plasmid (construct) can be used in a circular state or in a linear form, but is usually in a form that does not destroy the structural gene region and the expression control region such as a promoter. It is better to introduce it in a straight line.
- a transgenic mouse can be produced by transplanting a fertilized egg into which a target gene has been introduced into a pseudopregnant female mouse and delivering a pup mouse.
- the method of introducing a target gene into animal cells includes a method of introducing into an ES cell, and a fertilized egg by transplanting a cell nucleus introduced into a cultured cell. It is also possible to use a method introduced into the above.
- a vector incorporating the target gene DNA is introduced into ES cells or cultured cells using techniques commonly used in the art such as electroporation and lipofection, and then positively selected with neomycin, puromycin, etc.
- the desired introduced cell can be obtained.
- ES cells are injected into mouse embryo placental cysts or 8-cell stage embryos by microinjection or the like. Nuclear transfer can be performed by injecting a cell into which a target gene DNA has been introduced into a fertilized egg from which the nucleus has been removed, and fusing the cell with electrical stimulation.
- Embryo placental vesicles or 8-cell stage embryos into which ES cells have been injected as described above are transplanted directly into the foster tube of the foster parent, or those that have been cultured for one day and developed to the blastocyst are transplanted into the foster mother's uterus.
- a temporary parent is bred and a pup mouse is born, and a transgenic animal (chimeric animal) into which the target gene has been introduced can be obtained.
- genomic DNA in somatic cells is extracted from a part of the mouse tissue (for example, the tip of the tail), and PCR or Southern blotting is performed. Can be done by law. For example, it is possible to confirm whether or not the pup mouse holds the transgene by performing a PCR reaction using ex Taq enzyme using a genomic DNA prepared from the tail of the mouse as a template and an appropriate primer.
- transgenes are maintained in all cells, but whether or not this gene is expressed in the target tissue is expressed as RNA by RT-PCR. It can be confirmed by examining. Furthermore, whether or not the transgene is produced as a protein can be confirmed by immunoblotting using an antibody against the protein or its partial peptide.
- a primary transgenic animal Assuming that an individual in which the transgene is overexpressed as described above is a primary ( founder), when this primary transgenic animal is mated with a normal animal, a heterozygous animal (F1) A homozygous animal (F2) is obtained by crossing the heterozygotes. Furthermore, this target gene can be stably retained in the germ line by mating with animals of the same species and subcultured in a normal breeding environment.
- the IP3R binding protein according to the present invention can be identified by co-purifying a mouse organ tissue, for example, liver tissue, in which the target gene produced by the above method is overexpressed.
- a co-purification method for example, an immunoprecipitation method using an antibody against the target protein may be used. Thereby, it can be confirmed that the endogenous protein and the endogenous IP3R are bound to each other from a protein extract derived from a mouse biological tissue or a cell line.
- transgenic non-human mammal containing the IP3R binding protein and the IP3R binding protein according to the present invention will be described in more detail by way of examples, but the present invention is not limited in any way by the following examples, It should also be understood that the following examples are not intended to be limiting in any way. In addition, it should be understood that the present invention includes all variations and modifications that can be easily inferred from the present specification and the following examples.
- the entire length of the mouse KRAP coding region was inserted into the XhoI-BglII portion of the pCAGGS vector (Gene 1991, Niwa H et al.) To create an expression construct.
- the restriction enzyme XhoIho recognition sequence and mouse KRAP 5 'sequence 10b were added to the 5' portion of the coding region, and the HA tag, stop codon and restriction enzyme BamHI recognition sequence were added to the 3 'portion.
- the forward primer and reverse primer used were as follows, and an insert was prepared by PCR reaction using LA-taq enzyme (Takara) using mouse KRAP® cDNA registered in Accession No. AB120565 as a template.
- mice KRAP coding region was cloned into pcDNA / V5 / GW / D-TOPO vector (Invitrogen) with the HA tag added to the C terminus to prepare an expression construct.
- pcDNA / V5 / GW / D-TOPO vector Invitrogen
- HA tag added to the C terminus to prepare an expression construct.
- an insert was prepared by PCR reaction using LA-taq enzyme (Takara) ⁇ ⁇ ⁇ using mouse KRAP cDNA ⁇ registered in accession number AB120565 ⁇ ⁇ as a template.
- the deletion mutant consists of amino acid residues 988-1252 (coiled coil region only), amino acid residues 1-942, amino acid residues 1-318, amino acid residues 1-236, amino acid residues 1-218, and amino acid residues of mouse KRAP.
- DNA fragments encoding the region of the group 1-199 were prepared by PCR and cloned into the cloning site SalI-NotI of the pCMV-HA vector (Clontech).
- the N-terminal HA tag contained in the vector and the reading frame were connected together and constructed with a stop codon inserted at the C-terminus of the KRAP fragment.
- the point mutant is based on the KRAP coding region full-length construct created in the pcDNA / V5 / GW / D-TOPO vector described above, and each of the 202th phenylalanine and the 203rd phenylalanine counted from the first amino acid.
- F202A mutant and F203A mutant substituted with alanine and F202A / F203A mutant substituted with these two residues with alanine were prepared by KOD-Plus-Mutagenesis-Kit (TOYOBO).
- the used primers are as follows.
- Forward primer (for F202A) (SEQ ID NO: 13) 5'-gcatttaattcatcatcctttgccagaggc-3 ' Reverse primer (for F202A) (SEQ ID NO: 14) 5'-tctggaaggaattttagaagcaatatctg-3 ' Forward primer (for F203A) (SEQ ID NO: 15) 5'-gcaaattcatcatcctttgccagaggc-3 ' Reverse primer (for F203A) (SEQ ID NO: 16) 5'-aaatctggaaggaattttagaagcaat-3 ' Forward primer (for F202A / F203A) (SEQ ID NO: 17) 5'-gcagcaaattcatcatcctttgccagaggc-3 ' Reverse primer (for F202A / F203A) (SEQ ID NO: 18) 5'-tctggaaggaattttagaa
- FIG. 16 shows a schematic diagram of the mouse KRAP protein.
- the deletion mutant construct KRAP lacking the entire length, the coiled coil region (988-1252), and the coiled coil region is shown.
- (1-942) a deletion mutant construct KRAP (1-318, 1-236, 1-218, 1-199), in which the N-terminal region is shortened little by little, is shown at each N-terminal or C-terminal.
- HA tag is added.
- This example is an experiment for examining protein-protein interaction for determining a KRAP protein region important for binding to IP3R1.
- the binding ability of each KRAP deletion mutant and IP3R1 was examined.
- full-length or each deletion mutant was expressed together with IP3R1, and the protein extract was immunoprecipitated with anti-HA antibody to examine whether IP3R1 co-precipitated.
- the IP3R1 full-length-GFP expression plasmid and the KRAP full-length, deletion mutant, or point mutant expression plasmid prepared above were co-introduced into HEK293 cells using Lipofectamine LTX (Invitrogen), respectively, and cultured for 24 hours Later, homogenize in extraction buffer (50 mM Tris-HCl, pH 7.5, 140 mM NaCl, 0.5% NP40, 0.25 M NDSB-201 (Calbiochem), Protease inhibitor (Complete EDTA-free, Roche)), 15,000 rpm Therefore, the supernatant after centrifugation for 30 min was obtained.
- extraction buffer 50 mM Tris-HCl, pH 7.5, 140 mM NaCl, 0.5% NP40, 0.25 M NDSB-201 (Calbiochem), Protease inhibitor (Complete EDTA-free, Roche)
- This protein extract is immunoprecipitated with HA matrix (Roche) to coprecipitate the IP3R1-GFP protein bound to each KRAP protein, and the presence or absence of IP3R1-GFP protein in the precipitate is detected by Western blotting. did.
- a rabbit polyclonal antibody (Clontech) was used as the anti-GFP antibody.
- HCT116 cells In experiments with HCT116 cells, the presence or absence of co-precipitation of endogenous IP3R3 protein was examined without IP3R1-GFP protein being transfected.
- a mouse monoclonal antibody (BD transduction laboratory) was used as the anti-IP3R3 antibody.
- FIG. 17 shows the results of the total length of KRAP and the binding ability of each deletion mutant to IP3R1 in HEK293 cells according to this example.
- IP immunoprecipitation
- the amino acid region 1 to 218 retains the binding ability, whereas the amino acid region 1 to 199 loses the binding ability.
- the 19-amino acid long region of the 200-218th amino acid region is an important region for binding to IP3R1.
- IP3R is known to have a family consisting of genes different from types 1, 2 and 3, and the types expressed by cells / tissues are different. Therefore, KRAP protein is an IP3R other than type 1. And whether the binding mode is the same as that of type 1 or not. That is, only each deletion mutant was expressed in HCT116 cells, and the co-precipitation experiment of endogenous IP3R type 3 was performed in the same manner as in Example 5. As a result, the amino acid region of amino acids 1 to 218 at the N-terminal part of KRAP has binding ability with type 3 protein, whereas the amino acid region of amino acids 1 to 199 has no binding ability. From this, it became clear that it interacts with other types of IP3R in the mode of interaction with type 1 protein.
- FIG. 18 shows the results of the total length of KRAP and the binding ability of each deletion mutant to IP3R1 in HCT116 cells.
- FIG. 19a shows the amino acid sequence 202-220 of human KRAP (SEQ ID NO: 19) and the corresponding mouse KRAP amino acid sequence 200-218 (SEQ ID NO: 20) for the 19 amino acid long region. From the amino acid sequences of these mouse KRAP and human KRAP, it can be seen that the amino acid sequences are well conserved between the two species. In addition, the mutation site of the point mutant used in this example is shown below the amino acid sequences of mouse KRAP and human KRAP.
- a 6.2-kbp DNA fragment obtained by digesting a plasmid with BamHI was injected into a fertilized egg by microinjection.
- This fertilized egg was transplanted into the uterus of a pseudopregnant female mouse, and a pup mouse was born.
- Whether or not the transgene is retained in the offspring mouse was confirmed by PCR using ex Taq enzyme (Takara) using genomic DNA prepared from the tail of the mouse and using the DNA as a template with the following primers.
- the pups were mated according to the above method to establish a mouse strain as C57BL / 6J Jcl.
- FIG. 1 shows the results of PCR using ex Taq enzyme with the above primers using genomic DNA prepared from the established transgenic mouse tail as a template. From FIG. 1, 892 bp PCR products were amplified in the wild type (WT) and 636 bp in the transgenic mouse (TG), and it was revealed that each was a fragment of the expected size.
- Each tissue or cell of the generated transgenic mice was extracted with buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, protease inhibitor (Complete EDTA-free, (Roche) homogenized in a cage and centrifuged at 15,000 rpm for 30 minutes, and the supernatant was recovered to obtain a total extracted protein (totally lysate).
- the protein solution was developed by SDS-PAGE, transferred to a nitrocellulose membrane or PVDF membrane, and then the membrane was immunostained with an anti-HA antibody to detect the protein (FIG. 2).
- anti-HA ⁇ 3F10 clone, Roche
- Anti-KRAP J.Hum.Genet.2007, Fujimoto T. et al.
- Anti-IP3R1 Sigma
- Anti-HA -phospho-IP3R Ser 1756; Cell Signaling
- Anti-phospholipase C ⁇ Santa Cruz
- Anti-actin Sigma
- HRP-labeled antibody as secondary antibody
- ECL reagent GE Health Science
- FIG. 2 shows the results of Western blotting of a protein solution prepared from each tissue of a transgenic mouse and detection with an anti-HA antibody. From the results shown in FIG. 2, relatively strong KRAP-HA protein expression was confirmed in the liver, pancreas, skeletal muscle, and brown adipose tissue, while weak expression was confirmed in the kidney, spleen, and forebrain. In addition, when the amount of overexpression of KRAP in the liver was compared with that of the wild type, TG mice showed an expression enhancement of almost 4 times (FIG. 3).
- the localization of the overexpressed KRAP protein in the liver was examined by immunohistochemical staining with anti-HA antibody.
- Mouse livers were frozen in OCT compound (Sakura) and cryosections were made with cryostat. This section was fixed with 4% PFA, then blocked with a blocking solution (5% bovine serum, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tx-100), and Anti-HA (3F10 clone). , Roche).
- Alexa-Fluor-488-labeled anti-rat antibody Invitrogen
- Phalloidin-Alexa-Fluor-546 was used.
- liver extracts from KRAP transgenic (TG) mice were co-purified to identify KRAP binding proteins.
- Livers were collected from wild type (WT) mice and KRAP transgenic (TG) mice, and extracted buffers (50 mM Tris-HCl (pH 7.5), 140 mM NaCl, 0.5% NP40, 0.25 M NDSB-201 (Calbiochem), The mixture was homogenized in protease inhibitor (CompleteTAEDTA-free, Roche) and centrifuged at 18,500 rpm for 30 minutes to obtain a supernatant. Anti-HA Affinity Matrix (Roche) was added to the supernatant and mixed by inverting at 4 ° C for 5 hours.
- protease inhibitor CompleteTAEDTA-free, Roche
- FIG. 5 is an image of the HA-matrix binding fraction prepared from the livers of wild-type (WT) mice and transgenic (TG) mice developed by SDS-PAGE and silver-stained. As shown in FIG. 5, in the TG mouse, a band of KRAP protein to which HA-tag was added and a band co-purified with KRAP were detected.
- IP3R1 protein extraction of tissues liver, kidney, brown adipose tissue (BAT), pancreas) and cultured cells (HCT116, Hela) to clarify the presence or absence of physical interaction between endogenous KRAP protein and IP3R1 protein
- the immunoprecipitation product by anti-KRAP antibody was obtained from the product, and whether the precipitate contained IP3R1 protein was examined by Western blotting.
- Significant IP3R1 protein bands were detected in both tissue and cultured cell systems (FIGS. 8 and 9). Since no IP3R1 protein band was detected in the immunoprecipitation product of control IgG provided as a control, it is considered that the IP3R1 protein binding to the KRAP protein co-precipitated.
- HEK293 cells were transfected with KRAP with HA-tag and IP3R1 with GFP-tag, and immunostaining was performed.
- the cultured cells (HEK293 cells) were cultured in DMEM (high glucose, Invitrogen) supplemented with 10% bovine serum-containing penicillin / streptomycin / glutamine (Invitrogen) at 37 ° C. and 5% CO 2 .
- Cells were seeded on a collagen-coated cover glass, and KRAP-HA expression plasmid and IP3R1-GFP expression plasmid were introduced using Lipofectamine LTX (Invitrogen).
- a cDNA library was constructed by reverse transcription reaction with oligo-dT primer using mouse liver total RNA as a template. Using the cDNA as a template, the entire IP3R1 coding region was amplified by PCR with the following primers, and inserted into the NheI-SalI site of the pEGFP-N1 vector (Clontech) with the C-terminal GFP aligned.
- Intracellular calcium imaging was performed as follows.
- HEK293 cells were introduced with 1 nM stealth siRNA oligonucleotide by electroporation (using MicroPorator MP-100 (Digital Bio)), and penicillin, streptomycin, glutamine containing 10% bovine serum at a density of 30,000 cells in a 96-well plate.
- the cells were seeded in DMEM (high glucose, Invitrogen) supplemented with (Invitrogen) and cultured at 37 ° C. under 5% CO 2 condition.
- DMEM high glucose, Invitrogen
- 4 ⁇ M Fluo4 Invitrogen
- 5 ⁇ M Hoechst 33342 Invitrogen
- siRNA oligonucleotide used was used in a paper previously reported by the present inventor (J. Hum. Genet. 2007, Fujimoto T. et al.), And its sequence is as follows.
- Scramble RNA # 1 5'-G GAC GUA UAG UGU GAG AUA AAG AUU-3 '(SEQ ID NO: 31) 3'-C CUG CAU AUC ACA CUC UAU UUC UAA-5 '(SEQ ID NO: 32)
- KRAP # 2 5'-C CAG CUA GGU CUU ACG AAG UCG AAA-3 '(SEQ ID NO: 33) 3'-G GUC GAU CCA GAA UGC UUC AGC UUU-5 '(S
- FIG. 11 shows a representative example of the calcium indicator fluorescence signal change with time when the ATP final concentrations of 1 ⁇ M, 3 ⁇ M, and 10 ⁇ M were stimulated in KRAP gene expression-suppressed cells and non-suppressed cells.
- the KRAP-suppressed cells had lower fluorescence intensity than the control scramble ⁇ siRNA-treated cells.
- the upper side shows scramble siRNA-treated cells
- the lower side shows KRAP-suppressed cells.
- FIG. 12 shows data comparing the maximum fluorescence values after stimulation with ATP final concentrations of 1 ⁇ M, 3 ⁇ M and 10 ⁇ MATP.
- the maximum fluorescence value was obtained at 3 or 5.5 seconds after stimulation, a significant difference was observed during stimulation with ATP final concentrations of 1 ⁇ M and 3 ⁇ M, and was attenuated in KRAP-suppressed cells.
- the left side shows scramble siRNA-treated cells, and the right side shows KRAP-suppressed cells.
- FIG. 13 is a bar graph showing the maximum fluorescence increase (Mean peak amplitude) with the ratio of maximum fluorescence as 1 before ATP stimulation, and a significant difference was observed during 1 ⁇ M and 3 ⁇ M ATP stimulation, and KRAP suppression was observed. It was attenuated in the cells.
- the left side shows scramble siRNA-treated cells, and the right side shows KRAP-suppressed cells.
- FIG. 14 shows a bar graph of total fluorescence value after total stimulation (total fluorescence value after stimulation) showing the integration of fluorescence values after ATP stimulation (from 3 seconds after ATP stimulation to 25.5 seconds).
- total fluorescence value after stimulation shows the integration of fluorescence values after ATP stimulation (from 3 seconds after ATP stimulation to 25.5 seconds).
- a significant decrease in fluorescence value was observed in KRAP-suppressed cells under all conditions of 1 ⁇ M, 3 ⁇ M, and 10 ⁇ M ATP stimulation.
- the left side shows scramble siRNA-treated cells
- the right side shows KRAP-suppressed cells.
- FIG. 15 is a view showing suppression of KRAP expression in HEK293 cells by Western blot. As is clear from the figure, there was no significant change in the expression level of IP3R1 and the degree of phosphorylation of IP3R1. In addition, there was no change in the expression level of phospholipase C- ⁇ located upstream of signal transduction to IP3R1.
- IP3R regulates intracellular calcium ions, one of the second messengers, and is thought to function in various biological phenomena such as gene expression, cell life and death, nerve function, and secretory vesicle dynamics ( J. Neurochem. 2007, Mikoshiba K.).
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Abstract
Description
5'-gggctcgagcggcgcggccatgaaccgacccctgtcg-3'
リバースプライマー(配列番号10)
5'-gggggatcctcaagcgtaatctggaacatcgtatgggtagtggctgtcctgcttaggacc-3'
5'-caccatgaaccgacccctgtcg-3'
リバースプライマー(配列番号12)
5'-ctactaagatctctaagcgtagtctgggacgtcgtatgggtagtggctgtcctgcttagg-3'
5'-gcatttaattcatcatcctttgccagaggc-3'
リバースプライマー(F202A用)(配列番号14)
5'-tctggaaggaattttagaagcaatatctg-3'
フォワードプライマー(F203A用)(配列番号15)
5'-gcaaattcatcatcctttgccagaggc-3'
リバースプライマー(F203A用)(配列番号16)
5'-aaatctggaaggaattttagaagcaat-3'
フォワードプライマー(F202A/F203A用)(配列番号17)
5'-gcagcaaattcatcatcctttgccagaggc-3'
リバースプライマー(F202A/F203A用)(配列番号18)
5'-tctggaaggaattttagaagcaatatctg-3'
つまり、HCT116細胞に、各欠損変異体のみを発現させて、内在性のIP3Rタイプ3の共沈降実験を、実施例5と同様の方法で行った。その結果、KRAPのN末部分のアミノ酸番号1~218番目のアミノ酸領域はタイプ3タンパクとの結合能を有しているのに対し、アミノ酸番号1~199番目のアミノ酸領域は結合能が無かったことから、タイプ1タンパクとの相互作用様式で他のタイプのIP3Rとも相互作用することが明らかになった。図18に、HCT116細胞におけるKRAPの全長および各欠損変異体とIP3R1との結合能の結果を示す。
図19aは、上記19アミノ酸長の領域についてのヒトKRAP のアミノ酸配列202-220(配列番号19)ならびに対応するマウスKRAPのアミノ酸配列200-218(配列番号20)を示す。これらのマウスKRAPならびにヒトKRAP のアミノ酸配列から、2種間においてそのアミノ酸配列がよく保存されていることが分かる。また、マウスKRAPならびにヒトKRAP のアミノ酸配列の下には、本実施例で用いた点変異体の変異部位を示した。N末端から数えて202番目のフェニルアラニン(F)および203番目のフェニルアラニンを共にアラニン(A)に置換した変異体(Mouse KRAP F202A/F203A mutant;配列番号21)、またはどちらか一方を置換した変異体(Mouse KRAP F202A mutant:配列番号22またはMouse KRAP F203A mutant:配列番号23)である。図中、下線を引いたA(Ala)が野生型(wild)ではF(Phe)であった部分である。
5'-gtcacagagctaatgcggga-3'
リバースプライマー(配列番号25)
5'-cttcacactgcagttgagtc-3'および
リバースプライマー(配列番号26)
5'-ggcttcatgatgtccccat-3'
培養細胞(HEK293細胞)は、10%ウシ血清含有ペニシリン・ストレプトマイシン・グルタミン(インビトロジェン社)を添加したDMEM(high glucose, インビトロジェン社)中で37℃、5%CO2条件下で培養した。コラーゲンコートしたカバーガラス上に細胞を播き、リポフェクトアミンLTX(インビトロジェン社)を用いてKRAP-HA発現プラスミドおよびIP3R1-GFP発現プラスミドを導入した。24時間培養後、細胞を4%PFAで固定し、5%ウシ血清、20mM Tris-HCl(pH7.5)、150mM NaCl、0.1%Tx-100でブロッキング後、Anti-HA(3F10クローン、ロシュ社)およびAnti-GFP(クロンテック社)と反応させ、2次抗体としてAlexa Fluor 488標識抗ウサギ抗体(インビトロジェン社)とAlexa Fluor 546標識抗ラット抗体(インビトロジェン社)を用いて蛍光染色した。この免疫細胞染色を行った細胞(HEK293細胞)を共焦点レーザー顕微鏡で観察した結果、両タンパクの蛍光シグナルが良く一致しており共局在が観察された(図10)。
リバースプライマー(配列番号28):5'-ggggtcgactgggccggctgctgtgggttgacat-3'
KRAP #1, 5’-G GAG AAU GCU GAU AGU GAU AGA AUU-3’(配列番号29)
3’-C CUC UUA CGA CUA UCA CUA UCU UAA-5’(配列番号30)
Scramble RNA #1, 5’-G GAC GUA UAG UGU GAG AUA AAG AUU-3’(配列番号31)
3’-C CUG CAU AUC ACA CUC UAU UUC UAA-5’(配列番号32)
KRAP #2, 5’-C CAG CUA GGU CUU ACG AAG UCG AAA-3’(配列番号33)
3’-G GUC GAU CCA GAA UGC UUC AGC UUU-5’(配列番号34)
Scramble RNA #2, 5’-C CAU AGG UCU UAC GAA GUC GGC AAA-3’(配列番号35)
3’-G GUA UCC AGA AUG CUU CAG CCG UUU-5’(配列番号36)
IP3Rは、セカンドメッセンジャーの一つである細胞内カルシウムイオンを調節し、遺伝子発現や細胞の生死、神経機能、分泌小胞動態など多岐にわたる生命現象の調節に機能していると考えられている(J. Neurochem. 2007, Mikoshiba K.)。一方、KRAP欠損マウスは全身性のエネルギー恒常性異常、抗肥満・抗糖尿病の性質を示すことから(PLoS ONE 2009, Fujimoto et al.)、IP3Rの機能を正または負に調節する物質は、さまざまな生命現象を理解するための研究ツールとなるのみならず、疾患発症機序の理解や治療法の開発に繋がる可能性がある。
Claims (9)
- KRAPタンパク質(配列番号1~4)などのタンパク質と、イノシトール-1,4,5-トリスリン酸受容体(IP3R: 配列番号5~8)が結合していることを特徴とするイノシトール-1,4,5-トリスリン酸受容体結合タンパク質。
- 請求項1に記載のイノシトール-1,4,5-トリスリン酸受容体結合タンパク質であって、IP3Rが結合するタンパク質を構成するアミノ酸配列が、全長であっても、またはIP3Rが結合するアミノ酸部位を含むIP3R結合部位含有領域、つまりアミノ酸部分配列領域であることを特徴とするイノシトール-1,4,5-トリスリン酸受容体結合タンパク質。
- 請求項1または2に記載のイノシトール-1,4,5-トリスリン酸受容体結合タンパク質であって、該IP3R結合部位含有領域が、マウスKRAPタンパク質の場合、その200番目から218番目の19個のアミノ酸からなるアミノ酸配列領域であること、またはマウスKRAPタンパク質以外の動物種由来のKRAPタンパク質の場合、上記マウスKRAPタンパク質のアミノ酸配列領域に対応するアミノ酸配列領域であることを特徴とするイノシトール-1,4,5-トリスリン酸受容体結合タンパク質。
- 請求項1、2ないし3に記載のイノシトール-1,4,5-トリスリン酸受容体結合タンパク質であって、前記IP3R結合タンパク質または前記IP3R結合部位含有領域のアミノ酸一次構造もしくは高次構造情報に基づいて合成される構造模倣物または化合物であることを特徴とするイノシトール-1,4,5-トリスリン酸受容体結合タンパク質。
- 請求項1ないし4のいずれか1項に記載のイノシトール-1,4,5-トリスリン酸受容体結合タンパク質またはその結合部位含有領域を主成分とすることを特徴とする細胞内カルシウムイオン濃度調節剤。
- 請求項5に記載の細胞内カルシウムイオン濃度調節剤であって、該細胞内カルシウムイオン濃度調節剤が、IP3R機能調節剤、IP3R活性阻害剤またはIP3R活性化剤であることを特徴とする細胞内カルシウムイオン濃度調節剤。
- KRAPタンパク質とIP3RとのKRAP-IP3Rタンパク複合体もしくはIP3R結合部位含有領域を調製することまたはその結合能を検出することによってそのKRAP-IP3Rタンパク間の相互作用を阻害する物質をスクリーニングすることを特徴とするアッセイ方法。
- 請求項1ないし5のいずれか1項に記載のイノシトール-1,4,5-トリスリン酸受容体結合タンパク質またはその結合部位含有領域を含むことを特徴とするベクター。
- 請求項1ないし5のいずれか1項に記載のイノシトール-1,4,5-トリスリン酸受容体結合タンパク質またはその結合部位含有領域を含むことを特徴とするトランスジェニック非ヒト哺乳動物。
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US13/496,314 US20120180150A1 (en) | 2009-09-29 | 2010-09-28 | Inositol 1,4,5-triphosphate receptor-binding protein and its non-human transgenic mammalian animals |
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2001075067A2 (en) * | 2000-03-31 | 2001-10-11 | Hyseq, Inc. | Novel nucleic acids and polypeptides |
JP2004008216A (ja) * | 2002-03-22 | 2004-01-15 | Research Association For Biotechnology | 新規な全長cDNA |
WO2007010999A1 (ja) * | 2005-07-21 | 2007-01-25 | Japan Health Sciences Foundation | Krap遺伝子変異による脂質代謝および糖代謝改変動物、改変方法および改変剤 |
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2010
- 2010-09-28 WO PCT/JP2010/066864 patent/WO2011040417A1/ja active Application Filing
- 2010-09-28 JP JP2011534254A patent/JPWO2011040417A1/ja not_active Withdrawn
- 2010-09-28 US US13/496,314 patent/US20120180150A1/en not_active Abandoned
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WO2001075067A2 (en) * | 2000-03-31 | 2001-10-11 | Hyseq, Inc. | Novel nucleic acids and polypeptides |
JP2004008216A (ja) * | 2002-03-22 | 2004-01-15 | Research Association For Biotechnology | 新規な全長cDNA |
WO2007010999A1 (ja) * | 2005-07-21 | 2007-01-25 | Japan Health Sciences Foundation | Krap遺伝子変異による脂質代謝および糖代謝改変動物、改変方法および改変剤 |
Non-Patent Citations (4)
Title |
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AKIHIRO FUNAKOSHI ET AL.: "Suigai Bunpitsu Kino no Saisei o Mezashita Kiso Kenkyu", NANJISEI SUISHIKKAN NI KANSURU CHOSA KENKYU HEISEI 19 NENDO SOKATSU-BUNTAN KENKYU HOKOKUSHO, 31 March 2008 (2008-03-31) * |
FUJIMOTO T ET AL.: "Altered energy homeostasis and resistance to diet-induced obesity in KRAP-deficient mice", PLOS ONE, vol. 4, no. 1, 21 January 2009 (2009-01-21), pages E4240 * |
FUJIMOTO T ET AL.: "Analysis of KRAP expression and localization, and genes regulated by KRAP in a human colon cancer cell line", J. HUM. GENET., vol. 52, no. 12, 2007, pages 978 - 984, XP019545319, DOI: doi:10.1007/s10038-007-0204-8 * |
INOKUCHI J ET AL.: "Deregulated expression of KRAP, a novel gene encoding actin-interacting protein, in human colon cancer cells", J. HUM. GENET., vol. 49, no. 1, 2004, pages 46 - 52, XP003002988 * |
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