WO2018062392A1 - ダウン症モデルラット及びその作製法 - Google Patents
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Definitions
- the present invention relates to a novel model rat having symptoms of Down syndrome and a method for producing the same.
- Down syndrome (also referred to as “Down syndrome”) is a congenital disease caused by trisomy chromosome 21 in the human development process, such as mental retardation, cardiac malformation, early-onset Alzheimer's disease, childhood leukemia, immune system abnormalities, etc. It is known to produce an impaired phenotype. Furthermore, Down's syndrome is a chromosomal abnormality, so there is no cure, and little is known about the mechanism by which various disorders of Down's syndrome occur. Furthermore, regarding the phenotype of Down syndrome patients, it is not clear what gene (group) corresponds to the symptoms seen in Down syndrome, that is, the correlation between phenotype and genotype.
- Ts1Cje mouse and the Ts65Dn mouse are model mice having the chromosome 16 of the reconstructed mouse (Non-patent Document 1, Non-patent Document 2, Non-patent Document 3).
- Ts65Dn mice were produced by irradiating the testes of male mice, then mating those mice and selecting trisomy mice with chromosomal rearrangements.
- Down's syndrome model mice containing human chromosome 21 are produced, and that they exhibit behavioral abnormalities and cardiac abnormalities observed in Down's syndrome (Non-patent Documents 4 and 5).
- These mice were injected with mouse embryonic stem (ES) cells into which human chromosome 21 was introduced into mouse 8-cell embryos or blastocyst embryos, and then transplanted into the uterus. It was selected from among them.
- ES mouse embryonic stem
- An object of the present invention is to provide a Down syndrome model rat and a production method thereof.
- Non-Patent Document 5 As described above, it is known that production of Down syndrome model rats is very difficult unlike model mice. The reason is that rat ES cells capable of transmitting offspring could not be obtained. Also, as in Non-Patent Document 5 described above, even if human chromosome 21 is introduced into rat ES cells, injected into an early embryo, and transplanted into the uterus, a Down syndrome model rat capable of transmitting offspring is produced. Is difficult. Furthermore, Down's syndrome model mice produced by the method described in Non-Patent Document 4 did not always correspond to symptoms seen in humans.
- the present inventors have made full use of a technique combining rat ES cell (male strain) production, micronucleus cell fusion (MMCT) method, circular sperm injection (ROSI) method, etc.
- the present invention was completed for the first time by producing a model rat with Down syndrome capable of transmitting offspring.
- a Down syndrome model rat wherein a rat gene homologous to at least one gene on human chromosome 21 or a fragment thereof is trisomy and can be transmitted to offspring.
- a Down's syndrome model rat characterized in that at least one gene on a chromosome or a fragment thereof and an endogenous rat gene homologous to the gene are added and are trisomy and can be transmitted to offspring.
- the Down's syndrome model rat according to any one of (2) to (5), wherein the human chromosome 21 or a fragment thereof or the exogenous rat chromosome or a fragment thereof contains a DNA encoding a fluorescent protein.
- Step introducing the ES cell into a rat blastocyst stage embryo or an 8-cell stage embryo,
- the round sperm cells obtained from the male chimeric rat obtained were micro-fertilized into the ovum of the rat by the intra-ovum circular sperm injection method (ROSI), or the intra-ocular extended sperm cell injection method ( A step of microfertilizing the extended sperm cells of the male chimera rat and the egg of the rat by ELSI), or microfertilization of the sperm of the male chimera rat and the egg of the rat by the intracytoplasmic sperm injection method (ICSI) to produce a progeny rat And a method comprising the step of selecting, from the above-mentioned progeny rat, a human chromosome 21 or a fragment thereof or a progeny-transmittable rat carrying an exogenous rat
- the present invention provides a Down syndrome model rat capable of transmitting offspring.
- the rat is useful as a Down syndrome model animal because human chromosome 21 or a fragment thereof introduced therein or an exogenous rat chromosome or a fragment thereof is maintained extremely stably. This is useful for elucidating the mechanism.
- FIG. 1 a shows a schematic diagram of the procedure of Example 1.
- FIG. 1b shows hChr. Partial structure of 21-loxP allele, EGFP introduction vector (I-EGFP-I-loxP-3′HPRT), and modified human chromosome 21 (hChr.21-loxP) in which Cre-loxP recombination was performed by the vector -EGFP) allele partial structure.
- CHO represents Chinese hamster ovary cells, hChr.
- 21 represents human chromosome 21
- EGFP represents DNA encoding fluorescent protein
- HPRT or hprt represents hypoxanthine phosphoribosyltransferase gene
- Hyg represents hygromycin resistance gene
- TK represents thymidine kinase gene .
- the results of a mono-color FISH analysis of rat ES (hChr.21-loxP-EGFP) clones using human Cot-1 DNA as a probe are shown.
- the arrow indicates hChr.
- the presence of 21-loxP-EGFP is indicated.
- a summary of the production results of chimeric rats produced from rat ES (hChr.21-loxP-EGFP) is shown.
- GT indicates the number of male individuals with germline transmission (germline transmission).
- the hair color contribution ratio of ES cells, presence / absence of GFP positivity in whole body, presence / absence of tubule GFP positivity in a part of chimera rat male individuals (40% or more chimera) prepared from rat ES (hChr.21-loxP-EGFP) are shown. It can be seen that only one tubule GFP positive (# 1753-2) was obtained.
- the bright-field and GFP fluorescence photographs of testis in chimeric individual numbers # 1753-3-2 and # 1753-3 are shown. It can be seen that a part of # 1753-3 tubule is GFP positive (indicated by an arrow).
- the mono-color FISH analysis result (karyotype analysis result) of the blood culture cell of TC (hChr.21-loxP-EGFP) rat when using human Cot-1 DNA as a probe is shown.
- hChr. 21 represents human chromosome 21.
- the measurement result of the GFP positive rate (%) by the FCM analysis in the peripheral blood of TC (21HAC2) mouse and TC (hChr.21-loxP-EGFP) rat is shown.
- the bright field and GFP fluorescence field in each tissue of TC (hChr.21-loxP-EGFP) rat are shown. In each panel of the fluorescent field, the white shining part is GFP positive. Numbers indicate exposure time.
- Control represents a normal rat control
- DS represents a Down syndrome model rat. *, **, and *** indicate P ⁇ 0.05, P ⁇ 0.01, and P ⁇ 0.001, respectively.
- the result of the open field test (center dwell time (seconds) and total travel distance (meters)) is shown.
- Control represents a normal rat control
- DS represents a Down syndrome model rat.
- Control represents a normal rat control
- DS represents a Down syndrome model rat.
- the Down's syndrome model rat of the present invention is produced by producing rat ES cells (male strain), micronucleus cell fusion (MMCT), round sperm injection (ROSI) or intracytoplasmic sperm injection (ICSI) or ovum. It can be produced by a method that combines inwardly extended sperm cell injection (ELIS) and selection.
- rat ES cells male strain
- MMCT micronucleus cell fusion
- ROSI round sperm injection
- ICSI intracytoplasmic sperm injection
- ovum ovum
- the Down syndrome model rat capable of transmitting offspring of the present invention can be prepared as follows.
- the method comprises microcells containing male chromosome 21 or fragments thereof, or exogenous rat chromosomes or fragments thereof in which a rat gene homologous to human chromosome 21 or fragments thereof is present, and male rat ES cells.
- Fusing by the micronucleus cell fusion method thereby containing human chromosome 21 or a fragment thereof or exogenous rat chromosome or fragment thereof, and comprising at least one of said human chromosome 21 or fragment thereof or exogenous rat chromosome or fragment thereof
- a step of producing a trisomy ES cell by adding a gene and an endogenous rat gene homologous to the gene; introducing the ES cell into a rat blastocyst stage embryo or an 8-cell stage embryo;
- a process for producing a chimeric rat by transplanting it into the uterus of a foster parent, and a circular spermatozoon obtained from the obtained male chimeric rat Of the male chimeric rat and the egg of the rat by microinsemination by the circular sperm injection (ROSI) method in the ovum, or the extended sperm cell injection in the ovum (ELIC) method, or the egg cytoplasm.
- ROSI circular
- ICSI internal sperm injection
- the human chromosome 21 or a fragment thereof or an exogenous rat chromosome or a fragment thereof is a selectable marker gene for selecting the chromosome, for example, a DNA encoding a fluorescent protein (“ "Fluorescent gene").
- the Down syndrome model rat of the present invention is a Down syndrome model rat characterized in that a rat gene homologous to at least one gene on human chromosome 21 or a fragment thereof is trisomy and is capable of offspring transmission. May be.
- human chromosome 21 or a fragment thereof means a size of about 10 Mb to about 34 Mb of a substantially complete human chromosome 21, including a centromere, unless otherwise specified.
- substantially complete human chromosome 21 preferably refers to intact (ie, complete) human chromosome 21, but in the course of chromosome introduction or in the nucleus of a model rat. Means human chromosome 21 which may contain minor alterations in the base sequence that may occur accidentally (ie, do not affect the Down syndrome phenotype).
- a chromosome fragment having a size of about 10 Mb to about 34 Mb including the centromere of the complete human chromosome 21 the chromosome fragment affects the region for retaining the function as a chromosome and the phenotype of Down syndrome. And a long arm region.
- the “region for retaining a function as a chromosome” includes a telomere region, all or part of a long arm region, and the like in addition to a centromere region.
- the chromosomal fragment is a chromosome fragment of the complete human chromosome 21 having a size of about 10 Mb to about 34 Mb including centromere, preferably about 20 Mb to about 34 Mb, more preferably about 30 Mb to about 34 Mb. Genomic decoding of complete human chromosome 21 (M. Hattori et al., Nature 2000; 405: 311-319) has been performed, and it is known that there are at least 225 genes in about 34 Mb. .
- the foreign gene or foreign DNA is preferably as described above.
- exogenous rat chromosome or fragment thereof in which a rat gene homologous to human chromosome 21 or a fragment thereof is present refers to a rat gene homologous to a gene on human chromosome 21 or a fragment thereof.
- Exogenously introduced into the cell nucleus including a rat chromosome or fragment thereof exogenously introduced into the cell nucleus, more preferably a rat gene homologous to a gene on the Down Syndrome Critical Region (DSCR) refers to rat chromosomes or fragments thereof.
- This rat chromosome or fragment thereof is a substantially complete rat chromosome, a complete rat chromosome fragment of at least about 10 Mb including centromeres, Alternatively, the substantially complete rat chromosome or a variant thereof containing a foreign gene or DNA in the nucleotide sequence thereof, or the substantially complete rat chromosome, the chromosome fragment or the variant thereof It is used to indicate either a variant in which rat centromere is replaced with mouse centromere (for example, mouse centromere of mouse 11 or chromosome 16).
- the Down syndrome model rat characterized in that the rat gene homologous to at least one gene on human chromosome 21 or a fragment thereof relating to the present invention is trisomy and is capable of offspring transmission, has been described above.
- the rat gene homologous to at least one gene on human chromosome 21 or a fragment thereof relating to the present invention is trisomy and is capable of offspring transmission.
- at least one, preferably a plurality of rats are also included in which genes are trisomy genetically due to double inclusion of genes on endogenous rat chromosomes. Duplication of genes on the chromosome is described in, for example, L.A. E. Olson et al. , Science 2004, 306: 687-690.
- Rat ES cells Male line
- Rat ES cells were prepared in the same manner as mouse ES cells (MJ Evans and MH Kaufman, Nature 1981; 292 (5819): 154-156).
- a cell line having pluripotency and self-replication ability established from the inner cell mass of rat blastocyst stage embryo or 8-cell stage embryo.
- rat blastocysts in which egg zona pellucida has been lysed are cultured on a mouse embryonic fibroblast (MEF) feeder using a medium containing leukemia inhibitory factor (LIF), and blastocysts 7-10 days later
- LIF leukemia inhibitory factor
- the outgrowth formed from is dispersed, transferred onto a MEF feeder and cultured, and ES cells appear after about 7 days.
- rat ES cells for example, Kawaharada et al. , World J Stem Cells 2015; 7 (7): 1054-1063.
- male strain rat ES cells there are female and male strains of ES cells.
- male strain rat ES cells more preferably male strain rat ES cells prepared from hybrid rats, are often used.
- the ROSI method, and the fluorescence selection method By using the cells, the ROSI method, and the fluorescence selection method, a model rat capable of transmitting offspring can be obtained.
- Male ES cells can be selected by analyzing the XY karyotype of the produced ES cell line using an XY chromosomal probe (for example, available from Chromosome Science Labo Inc.).
- XY chromosomal probe for example, available from Chromosome Science Labo Inc.
- Non-Patent Documents 4 and 5 female strain ES cells are used when producing Down syndrome model mice, but when male strain ES cells are used, establishment of mice capable of transmitting offspring failed. (Non-Patent Document 5). From this point of view, it can be seen that various trials and errors were repeated for the production of the Down syndrome model rat of the present invention. That is, in fact, such a model rat has not been established.
- iPS Artificial pluripotent stem cells are known as stem cells similar to ES cells (K. Takahashi and S. Yamanaka, Cell 2006; 126 (4): 663-676; K. Takahashi et al., Cell 2007; 131 (5): 861-872).
- Rat iPS cells have also been prepared in a similar manner (W. Li et al., Cell Stem Cell 2009; 4: 16-19; S. Hamanaka et al., PLoS One 2011; 6: e22008). That is, W.W. According to Li et al.
- WB-F344 rat hepatic endothelial cells were transduced with Oct4, Sox2 and Klf4 via retrovirus and then on a MEF feeder cell in a conventional mouse ES cell medium containing LIF.
- Rat iPS cells are produced by culturing. Therefore, in the present invention, rat iPS cells can be used as an alternative to ES cells.
- micronucleus cell fusion The micronucleus cell fusion method is also referred to as microcell-mediated chromosome transfer (MMCT).
- a microcell is a cell that is produced by enucleating a micronucleated cell obtained by micronucleating a cell (donor cell) and containing a small nucleus containing a small amount of cytoplasm and one or a small number of chromosomes.
- This microcell makes it possible to introduce a megabase-sized nucleic acid, such as a single chromosome or chromosome fragment, into the cell by fusing with the recipient cell.
- Such a fusion method is generally called a microcell fusion method or a micronucleus cell fusion method (for example, JP-A-2011-177145).
- the donor cell that can induce microcells is an animal cell, preferably a mammalian cell, and the cell type is not limited as long as the microcell can be induced.
- Animal cells include all forms of cells such as primary cells, established cells, passaged cells, cultured cells, somatic cells, stem cells, and the like.
- Animal cells include, for example, invertebrate cells such as insect cells, mammalian cells such as human cells and rodent cells, avian cells, amphibian cells, reptile cells, fish cells, and other vertebrates. Derived cells, preferably rodent cells (CHO cells, mouse A9 cells, etc.).
- a selection marker gene for example, a fluorescent gene
- a variant of human chromosome 21 for example, a region such as a centromere region of human chromosome 21
- a vector having a selectable marker gene (for example, a fluorescent gene) and a loxP sequence, and a Cre expression vector can be co-introduced in advance into a donor cell having a loxP sequence.
- a selection marker gene for example, a fluorescent gene
- human chromosome 21 having a selectable marker gene is useful for confirming whether or not a germline transmission has occurred using a selective characteristic (for example, a fluorescent characteristic).
- the fluorescent gene is DNA encoding the fluorescent protein, and examples of the fluorescent protein include GFP, EGFP, YFP, EYFP, Venus, CFP, ECFP, Keima, DsRed, and the like, but are not limited thereto.
- the amino acid sequence of the fluorescent protein and the base sequence of the DNA encoding the fluorescent protein are available from gene banks such as GenBank and known literature. For example, the amino acid sequence and base sequence of EGFP are described in GenBank accession number LC008492 (S.
- GFP and YFP are described in, for example, WO2012 / 063897A1.
- exogenous rat chromosome the same operation as in the case of human chromosome 21 can be performed.
- the micronucleus cell fusion method is a technique that enables transfer of a large nucleic acid of 1 Mb or more such as a single or a small number of chromosomes or fragments thereof from a donor cell to a recipient cell.
- the method includes a first step of micronucleating donor cells, a second step of enucleating micronucleated cells, a third step of isolating microcells, a fourth step of fusing microcells and recipient cells, and survival.
- Micronucleation of donor cells can be performed by culturing animal cells for a long time in a medium containing a micronucleus cell inducer such as colcemid.
- the micronucleus cell inducer has the ability to induce chromosome decondensation and nuclear membrane re-formation.
- Concentration of micronucleated cells inducing agent is not limited if fine nucleation occurs, for example, in the case of colcemid, recipient cells about 5 ⁇ 10 6 per about 0.01 [mu] g / ml ⁇ about 1 [mu] g / ml, preferably 0. 05-0.5 ⁇ g / ml.
- micronucleation cells containing micronuclei containing a small amount of cytoplasm and one or a few chromosomes, ie microcells, are formed from donor cells. Culturing uses the culture conditions of the donor cells, and animal cell media are generally used as media. Examples of the medium for animal cells include Eagle's medium (MEM), Eagle's minimum essential medium (EMEM), Dulbecco's modified Eagle medium (DMEM), Ham's F12 medium, and the like. Fetal bovine serum (FBS), alternative serum (Stem Sure (R) Serum Replacement, etc.) and the like may be added to the medium.
- the temperature is room temperature to about 37 ° C., and the culture time is suitably about 40 to 80 hours.
- Enucleation of micronucleated cells is performed using cytochalasin B.
- a culture solution containing micronucleated cells is placed in a centrifuge tube, cytochalasin B is added at a concentration of about 10 ⁇ g / ml, and centrifugation is performed at 34 ° C. at about 11,900 ⁇ g.
- the sedimented microcell is suspended and collected in a serum-free medium.
- the microcell can be purified by ultrafiltration. Three types of membranes having a pore diameter of 8 ⁇ m, 5 ⁇ m and 3 ⁇ m are prepared and filtered in order.
- the purified microcell is layered on the recipient cell that has been cultured before becoming completely confluent.
- Microcell fusion cells can be obtained by a technique such as selecting drug-resistant strains.
- the fusion is carried out by a method of polyethylene glycol (PEG) method, mouse leukemia virus (MLV) method, retro method, etc. (T. Suzuki et al., PLOS ONE, DOI: 10.1371 / journal. Pon. 0157187 (2016)). , MV method (M. Katoh et al., BMC Biotechnology 2010, 10:37) and the like.
- the retro method is a method in which microcells and recipient cells are fused using R-peptide-deleted Env (Env ⁇ R) derived from homotrophic or ecotropic MLV, and is the most rodent cell. It is an efficient method.
- the MV method is a method for promoting microcell fusion using a hemagglutinin protein (MV-H) and a fusion protein (MV-F), which are measles virus fusogen, in advance, and the MV-H plasmid.
- MV-H hemagglutinin protein
- MV-F fusion protein
- microcells made from donor cells transformed with the MV-F plasmid are susceptible to cell-cell fusion with recipient cells due to the presence of fusogen expressed on the cell membrane surface.
- a human chromosome is introduced as a foreign nucleic acid into the donor cell in advance.
- the human chromosome moves to the microcell and is introduced into the recipient cell by microcell fusion.
- the recipient cell is transformed with a human chromosome.
- microcell fusion can be preferably used for transferring (or introducing) a nucleic acid having a size of 1 Mb or more, particularly a specific chromosome or chromosome fragment having a megabase size, from one cell to another.
- the chromosomal fragment is a fragment of human chromosome 21 or an exogenous rat chromosome fragment in which a homologous gene (s) is present, and contains about 10 Mb to Preferably it has a size of about 34 Mb.
- Microinsemination methods such as round sperm injection (ROSI) in ova.
- Rat ES cells were introduced into blastocyst stage embryos and transplanted into temporary parents' uterus to produce chimeric rats, and offspring were obtained by mating from chimeric rats.
- a fluorescent gene expression positive rat that is, a progeny transmission rat was not obtained.
- the possibility of offspring transmission by the ROSI method was examined, and a human chromosome 21 or a fragment thereof or an exogenous rat chromosome or fragment thereof capable of stably transmitting the target offspring is obtained with a very low probability. I was able to.
- round sperm cells are aspirated into the pipette from the seminiferous tubules taken out from the testis of the chimeric rat (male), and the nucleus and cytoplasm are separated in the pipette. Thereafter, this is injected into a rat egg and microinsemination (C. Tsurumaki et al., J. Mamm. Ova Res. 2009; 26: 86-93 (Jp)).
- round sperm cells can be obtained from rats by ejaculation.
- a pure strain or female rat (or male rat) retaining human chromosome 21 or a fragment thereof or exogenous rat chromosome or fragment thereof is used.
- a hybrid preferably a hybrid male rat (or female rat) can be crossed to obtain a karyotype-stable trisomy rat in which human chromosomes are retained in about 80 to 90% or more of the cell nuclei in various rat tissues.
- ICSI intracytoplasmic sperm injection
- ELIS in-ovum extended sperm cell injection method
- human chromosome 21 or fragments thereof various tissues (for example, brain Thymus, heart, lung, liver, spleen, kidney, small intestine, testis, etc.) are analyzed by FISH (Fluorescence In Situ Hybridization) method, and human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof is retained or retained. Rate measurement, light / dark test, open field test, elevated plus maze test, etc.
- FISH Fluorescence In Situ Hybridization
- RNAISH fluorescently labeled specific oligonucleotide probe is used to hybridize with a gene on human chromosome 21 or a fragment thereof, and the chromosome is detected using a fluorescence microscope.
- the rat selected by such a method is determined to be a Down syndrome model rat having a Down syndrome phenotype, stably having human chromosome 21 or a fragment thereof, and capable of transmitting offspring. can do.
- progeny transmittable means that human chromosome 21 or a fragment thereof as defined above or an exogenous rat chromosome or a fragment thereof is stably transmitted to progeny (offspring) by germline transmission.
- the specific manufacturing procedure can include the following steps, for example.
- Step 1 In a rodent cell such as a CHO cell, the EGFP gene is introduced into the human chromosome 21 or a fragment thereof as defined above, or an exogenous rat chromosome or a fragment thereof retaining loxP in the centromere region using the Cre-loxP system. Whether the human chromosome 21 region or a fragment thereof or the exogenous rat chromosome or a fragment thereof is retained is confirmed by the PCR method and the FISH method.
- Step 2 The modified human chromosome 21 or fragment thereof or exogenous rat chromosome or fragment thereof is introduced into rat ES cells (male strain) by the micronucleus fusion method, and human chromosome 21 or fragment region thereof or exogenous rat chromosome Alternatively, whether the fragment region is retained is confirmed by PCR and FISH methods.
- Step 3 The rat ES cells are injected into a blastocyst stage embryo and transplanted into a foster parent's uterus to produce a chimeric rat.
- Step 4 Microinsemination by circular sperm cell injection method, intracytoplasmic sperm injection method or in ovum extended sperm cell injection method using circular sperm cells, sperm or extended sperm cells from the above chimeric rat male and ova from rat female
- Progeny transmission rats are produced by using the tail DNA and cells, and it is confirmed by PCR method and FISH method whether human chromosome 21 or its fragment or exogenous rat chromosome or its fragment is retained.
- Step 5 Trisomy rats are bred by mating.
- Step 6) The GFP positive rate in blood cells is measured by FCM analysis.
- Step 7 Fluorescent gene expression positive rate in blood cell lineage is measured by flow cytometry (FCM) analysis using various antibodies specific to blood cell lineage.
- Step 8 Each tissue is collected, and the retention of human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof is measured by FISH analysis.
- Step 9 The gene expression on human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof in each tissue is confirmed by RT-PCR.
- Step 10 Comparison with normal rats is made by various behavioral analysis and brain and heart abnormality analysis.
- mice In general, in contrast to Down syndrome model mice, when injecting substances such as drugs into specific locations such as electrophysiological experiments and brainstem nuclei, the size of the rat brain is appropriate, and the amount of spontaneous activity in mice is high. While the results of tests such as cognitive functions are often unclear, rats may conduct sophisticated cognitive function tests for higher brain dysfunction because they have lower spontaneous activity and higher intelligence The advantages of using rats, such as being able to do so, are known.
- the Down's syndrome model rat of the present invention has never been produced in the past, and the reason for this is that it was very difficult to produce as described above, but the rat produced this time was introduced exogenously.
- Stably holding human chromosome 21 or a fragment thereof or rat chromosome or a fragment thereof (preferably one) in the nucleus, or introduced human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof At least one, preferably a plurality, of endogenous rat genes that are homologous to the gene are added to have a trisomy, have a Down syndrome phenotype, and are capable of offspring transmission.
- homologous refers to having about 70% or more sequence identity with respect to the base sequence of the mature gene, or having substantially the same biological function. Sequence identity refers to the percentage (%) of the number of matched bases relative to the total number of bases including the gap when the mature sequence of the human gene and the mature sequence of the rat gene are aligned and compared with the highest match rate. .
- sequence identity refers to the percentage (%) of the number of matched bases relative to the total number of bases including the gap when the mature sequence of the human gene and the mature sequence of the rat gene are aligned and compared with the highest match rate.
- an endogenous rat gene (2 copies) and an exogenous human gene ( 1 copy) or exogenous rat gene (1 copy) genetically trisomy.
- a model rat containing a human chromosome 21 fragment containing such a gene homologous region is genetically trisomy.
- a human chromosome 21 or a fragment thereof or an exogenous rat chromosome or a fragment thereof is stably retained in the cell nucleus.
- the human chromosome 21 or a fragment thereof as defined above or genes on an exogenous rat chromosome or a fragment thereof (for example, The presence of the genes described in Non-Patent Documents 4 and 5) can be confirmed by PCR analysis.
- the presence and retention of human chromosome 21 or fragments thereof or exogenous rat chromosomes or fragments thereof in rat blood cells and various tissues can be confirmed by FISH analysis or flow cytometry analysis.
- CD61 + / CD45RA ⁇ (NK cell-like), CD3 + / CD4 + (CD4 + T cell), CD3 + / CD8 + (CD8 + T cell), CD45R + (B cell) in any of peripheral blood, bone marrow, and spleen Like) Retention rate of human chromosome 21 or its fragment or exogenous rat chromosome in immune cells such as cells is 90% or more, preferably 95% or more, more preferably 98% or more.
- human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof in each tissue eg, brain, thymus, heart, lung, liver, spleen, kidney, small intestine, testis, bone marrow, etc. It is 90% or more, preferably 95% or more, and more preferably 98% or more.
- the Down syndrome model rat has at least one gene on complete or substantially complete human chromosome 21 or exogenous rat chromosome, or human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof.
- Human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof is stably retained in the cell nucleus so that a homologous endogenous rat gene forms trisomy.
- Progeny can be transmitted Human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof is transmitted to a progeny (offspring) by a germline transmission.
- human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof is retained extremely stably.
- Example 1 EGFP-loaded human chromosome 21 hChr. Production of 21-loxP-EGFP [A] hChr. Transfer of 21-loxP into CHO cells [A. 1] Micronucleus cell fusion and isolation of drug resistant clones In recipient cells, Kazuki et al. (Gene Therapy, 18: carrying a modified human chromosome 21 having a loxP sequence introduced in the vicinity of the long arm centromere of human chromosome 21: 384-393, 2011), DT40 (hChr.21-loxP) was used.
- DT40 hChr.21-loxP
- RPMI1640 medium supplemented with 20% FBS, 1% chicken serum, 10-4M 2-mercaptoethanol, 0.05 ⁇ g / ml colcemid
- the culture solution was replaced with 24 ml of serum-free DMEM medium, and 2 ml each was dispensed into 12 centrifuge 25 cm 2 flasks (Corning) previously coated with 100 ⁇ / ml poly L-lysine, and cultured at 37 ° C. for 30 minutes. Cells were attached to the bottom of the flask.
- cytochalasin B (10 ⁇ g / ml, sigma) solution pre-incubated at 37 ° C. was filled in the centrifuge flask, followed by centrifugation at 34 ° C., 8000 rpm for 1 hour.
- the microcell was suspended in serum-free DMEM medium and purified with 8 ⁇ m, 5 ⁇ m and 3 ⁇ m filters. After purification, the mixture was centrifuged at 1700 rpm for 10 minutes and suspended in 5 ml of serum-free DMEM medium.
- CHO HPRT ⁇
- CHO which is a CHO hprt deficient cell (obtained from Human Science Research Resource Bank (Osaka, Japan), registration number JCRB0218)
- the purified micronucleated cells were resuspended in 2 ml of a serum-free culture medium containing PHA-P (SIGMA), and gently seeded on CHO cells from which the culture supernatant [F12 medium supplemented with 10% FBS (Invitrogen)] was removed. .
- the plate was incubated at 37 ° C. for 15 minutes.
- PEG1000 (Wako) solution [5 g of PEG1000 was completely dissolved in serum-free DMEM medium and 1 ml of dimethyl sulfoxide (DMSO) was added and sterilized by filtration] was fused with 1 ml for exactly 1 minute.
- Serum-free culture medium (DMEM) was washed 4 times with 4 ml, and 5 ml of normal CHO cell culture medium was added and incubated overnight. After washing the cell surface twice with PBS ( ⁇ ), the cells were dispersed by trypsin treatment, seeded in 5 cell culture dishes having a diameter of 10 cm, G418 was added to 800 ⁇ g / ml, and selective culture was performed for 1 to 2 weeks.
- a total of 6 resistant colonies obtained by two micronucleus cell fusions were isolated and expanded, and the subsequent analysis was performed (clone name: CHO (HPRT ⁇ ; hChr.21-loxP)).
- PCR uses Perkin-Elmer's GeneAmp9600 as thermal cycler, Taq polymerase uses EX Taq (Takara Bio, Kyoto, Japan), and buffers and dNTPs (dATP, dCTP, dGTP, dTTP) are recommended. Used according to conditions. The temperature and cycle conditions were 35 cycles of heat denaturation at 93 ° C for 5 minutes followed by 93 ° C for 1 minute, 56 ° C for 1 minute, and 72 ° C for 1 minute. As a result of PCR, 6 clones out of 6 clones were positive in all primer sets, and the subsequent analysis was performed on these 6 clones.
- PCR analysis PCR was performed using the following primers to select recombinants using the genomic DNA of a HAT-resistant strain as a template, and it was confirmed whether GFP gene insertion occurred site-specifically.
- the primer sequences are shown below.
- TRANS L1 5'-tggaggccataaacaagaagac-3 '(SEQ ID NO: 15)
- TRANS R1 5'-ccccttgacccagaaattccA-3 '(SEQ ID NO: 16)
- PCR GeneAmp9600 manufactured by Perkin-Elmer is used as a thermal cycler, LA Taq (Takara Bio) is used as Taq polymerase, and 2 ⁇ GCI buffer and dNTPs (dATP, dCTP, dGTP, dTTP) are in accordance with the recommended conditions. Using. Temperature and cycle conditions were heat denaturation at 98 ° C. for 1 minute, followed by 30 cycles of 94 ° C. for 10 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 3 minutes. As a result of PCR, all 6 clones were positive, and the subsequent analysis was performed on these 6 clones.
- Example 2 hChr. Production of 21-loxP-EGFP-introduced rat ES cells hChr. In order to create a chimeric rat carrying 21-loxP-EGFP, the hChr. The CHO cells retaining 21-loxP-EGFP were introduced into rat ES cells by the micronucleus cell fusion method.
- Microcells were purified from CHO cells harboring 21-loxP-EGFP (CHO (hChr.21-loxP-EGFP) 1) and suspended in 5 ml of DMEM. About 10 7 rat ES cells were detached by trypsin treatment, washed 3 times with DMEM, suspended in 5 ml of DMEM, added to the centrifuged microcell, and centrifuged at 1250 rpm for 10 minutes to completely remove the supernatant.
- hChr Production of 21-loxP-EGFP-introduced rats
- a blastocyst stage embryo obtained by male-male mating of Crlj: WI rats (white, purchased from Nippon Charles River (Yokohama, Kanagawa, Japan)) was used.
- hChr As a result of transplanting 125 embryos injected with 21-loxP-EGFP-carrying ES clones (eg, Rat ES (hChr.21-loxP-EGFP) 2 obtained in Example 2 above) into 65 parents, 65 chimeric rats were transplanted. (A dark brown part is recognized in the fur color) was born. Of these, 53 were chimeric individuals whose coat color could be observed as colored (FIG. 3). That is, hChr. It was shown that the rat ES cell line carrying 21-loxP-EGFP retains the ability to form chimeras, that is, retains the ability to differentiate into normal tissues of rat individuals.
- 21-loxP-EGFP-carrying ES clones eg, Rat ES (hChr.21-loxP-EGFP) 2 obtained in Example 2 above
- GFP-positive circular sperm cells are injected into the ovum using the circular sperm cell intra-ocular injection method, and 40 embryos are temporarily parented. And 13 rats were produced. Among them, there were 5 GFP positive individuals. Furthermore, in 4 out of 5 animals, hChr. It was positive by PCR using the above primer for detecting the 21-loxP-EGFP region. Furthermore, the blood of the above individual was collected, and as a result of FISH analysis (Tomizuka et al., Nature Genet. 16: 133-143, 1997) using human Cot-1 DNA, using the chromosome specimen after culture.
- Example 4 Gene analysis of TC (hChr.21-loxP-EGFP) rat [A] Observation with stereofluorescence microscope About TC (hChr.21-loxP-EGFP) rat obtained above, brain, thymus, heart, lung When the liver, kidney, spleen, small intestine, skeletal muscle and testis were observed under stereoscopic fluorescence microscopy, GFP positivity was observed in all tissues, and the positive rate was 100%. A representative result is shown in FIG.
- FISH Fluorescence in situ hybridization
- the transmission rate was examined by mating males. From the F2 generation to the F6 generation, 309 pups were obtained, 181 were GFP-negative and 128 were GFP-positive individuals (transmission rate was 41.4%). That is, although the transmission rate is slightly lower than Mendelian inheritance law (theoretical value: 50%), hChr. It was confirmed that the 21-loxP-EGFP trait appeared at a frequency of 41.4%, and hChr. It was shown that the retention rate of 21-loxP-EGFP was 80% or more.
- PCR uses Perkin-Elmer's GeneAmp9600 as thermal cycler
- Taq polymerase uses EX Taq (Takara Bio (Kyoto, Japan)
- buffers and dNTPs dATP, dCTP, dGTP, dTTP
- the temperature and cycle conditions were 35 cycles of heat denaturation at 93 ° C for 5 minutes followed by 93 ° C for 1 minute, 56 ° C for 1 minute, and 72 ° C for 1 minute.
- Example 5 Behavioral analysis of TC (hChr.21-loxP-EGFP) rats
- A Light-dark traffic test Using TC (hChr.21-loxP-EGFP) rat strain, GFP-positive individuals were identified as Down syndrome model rats (hereinafter referred to as “Right syndrome”). , “DS”), and GFP-negative individuals were used as controls (hereinafter referred to as “Control”). Seven 10-week-old males were prepared for each group. In order to reduce the stress caused by touching the experimenter, habituation to humans (handling 5 minutes) was performed at the age of 9 weeks. Using these animals, a light-dark traffic test was conducted with reference to the method described on the RIKEN web page (http://ja.brc.riken.jp/lab/bmpp/SOPs/index_mc.html). .
- a light / dark cage for rats (LDK-R, Merquest) is set on the small animal behavior analyzer SCANET (MV-40, Merquest), and the light / dark cage is used by the software for small animal behavior analysis system (SCL-40, Merquest).
- SCANET dead band setting was 10.
- a daylight-colored light bulb was used, and the light intensity at the center of the bright box was set to 200 to 213 lux.
- Elevated plus maze test Six male TC (hChr.21-loxP-EGFP) rats at 10 weeks of age were prepared in each of the DS and control groups. Acclimatization to humans (handling 5 minutes) was performed at the age of 9 weeks, and the animals used for the light / dark traffic test and the open field test were also used for the elevated plus maze test.
- the elevated plus maze test was conducted as follows. In order to familiarize the animals with the behavior analysis room, the experiment was started one hour or more after the animals were moved from the breeding room to the behavior analysis room. Elevated cross labyrinth for rats with daylight-colored light bulb set to 39-42 lux at the center of the device (arm width 10 cm, arm length 50 cm, arm height 40 cm, neutral zone 10 cm x 10 cm, Muromachi Machine Stock Company).
- the animal's head was placed in a neutral zone with the closed arm in place, and 5-minute animal behavioral data was acquired with video tracking software (ANY-maze, Steeling).
- video tracking software ANY-maze, Steeling
- the open arm stay time tended to be low in DS (FIG. 14). From the above results, DS showed an increase in anxiety-like behavior.
- the Down syndrome model rat of the present invention can stably transmit offspring of human chromosome 21 or a fragment thereof or exogenous rat chromosome or a fragment thereof, and has refined cognition because it has symptoms of Down syndrome (behavioral abnormalities, etc.). Since functional tests can be performed, it is industrially useful as a model animal for developing therapeutic drugs and elucidating the onset mechanism.
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Abstract
Description
(1)ヒト21番染色体もしくはその断片上の少なくとも1つの遺伝子と相同なラット遺伝子がトリソミーである、かつ、子孫伝達可能であることを特徴とするダウン症モデルラット。
(2)ヒト21番染色体もしくはその断片、又はヒト21番染色体もしくはその断片と相同なラット遺伝子が存在する外因性ラット染色体もしくはその断片、を含み、当該ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片上の少なくとも1つの遺伝子と、当該遺伝子と相同な内因性ラット遺伝子とが加わりトリソミーである、かつ、子孫伝達可能であることを特徴とするダウン症モデルラット。
(3)上記ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片が、子孫ラットの組織内で80~90%以上の保持率で保持されている、上記(2)に記載のダウン症モデルラット。
(4)正常ラットと比較して、不安様行動又は記憶障害を含む行動異常を有する、上記(2)又は(3)に記載のダウン症モデルラット。
(5)上記ヒト21番染色体の断片又は外因性ラット染色体の断片が、セントロメア領域を含み、かつ10~34Mbのサイズを有する、上記(2)~(4)のいずれかに記載のダウン症モデルラット。
(6)上記ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片が、蛍光タンパク質をコードするDNAを含む、上記(2)~(5)のいずれかに記載のダウン症モデルラット。
(7)上記(2)~(6)のいずれかに記載の子孫伝達可能なダウン症モデルラットの作製方法であって、ヒト21番染色体もしくはその断片を含む、又はヒト21番染色体もしくはその断片と相同なラット遺伝子が存在する外因性ラット染色体もしくはその断片を含む、ミクロセルと雄系統のラットES細胞とを微小核細胞融合法により融合し、それによりヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片を含み、上記ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片上の少なくとも1つの遺伝子と、当該遺伝子と相同な内因性ラット遺伝子とが加わりトリソミーであるES細胞を作製する工程、上記ES細胞をラット胚盤胞期胚又は8細胞期胚に導入し、得られた胚をラット仮親の子宮に移植してキメララットを作製する工程、得られた雄キメララットから得た円形精子細胞をラットの卵子に卵子内円形精子注入法(ROSI)により顕微受精し、或いは卵子内延長精子細胞注入法(ELSI)により上記雄キメララットの延長精子細胞とラットの卵子を顕微受精し、或いは卵細胞質内精子注入法(ICSI)により上記雄キメララットの精子とラットの卵子を顕微受精し、子孫ラットを作製する工程、並びに、上記子孫ラットからヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片を保持する子孫伝達可能なラットを選別する工程を含む方法。
(8)上記ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片が、蛍光タンパク質をコードするDNAを含む、上記(7)に記載の方法。
(9)上記ヒト21番染色体の断片又は外因性ラット染色体の断片が、セントロメア領域を含み、かつ10~34Mbのサイズを有する、上記(7)又は(8)に記載の方法。
1.ダウン症モデルラットの作製
本発明のダウン症モデルラットは、ラットES細胞(雄系統)の作製、微小核細胞融合(MMCT)、卵子内円形精子注入(ROSI)もしくは卵細胞質内精子注入(ICSI)もしくは卵子内延長精子細胞注入(ELSI)、並びに選別を組み合わせた方法によって作製することができる。
(1)ラットES細胞(雄系統)の作製
ラットのES細胞は、マウスES細胞の場合と同様に(M.J.Evans and M.H.Kaufman,Nature 1981;292(5819):154-156)、ラット胚盤胞期胚又は8細胞期胚の内部細胞塊から樹立される、多分化能と自己複製能をもつ細胞株である。例えば、卵透明帯を溶解したラット胚盤胞を、白血病抑制因子(LIF)を含有する培地を用いてマウス胚性線維芽細胞(MEF)フィーダー上で培養し、7~10日後に胚盤胞から形成されるアウトグロウス(outgrowth)を分散し、これをMEFフィーダー上に移して培養し、約7日後、ES細胞が出現する。ラットES細胞の作製については、例えばK.Kawaharada et al.,World J Stem Cells 2015;7(7):1054-1063に記載されている。
微小核細胞融合法は、ミクロセル仲介染色体移入(Microcell-Mediated Chromosome Transfer;MMCT)とも称される。
ラットES細胞を胚盤胞期胚に導入し、仮親の子宮に移植してキメララットを作製し、キメララットから交配により子孫を得たところ、蛍光遺伝子発現陽性ラット、すなわち子孫伝達ラットが得られなかった。このため、ROSI法による子孫伝達の可能性を検討したところ、非常に低い確率で目的の子孫伝達可能なヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片を安定に保持するラットを得ることができた。
上記(3)で交配によって作製されたラットの中から子孫伝達可能なダウン症モデルラットを選別する方法は、蛍光遺伝子発現の陽性率を血液細胞や、末梢血、骨髄又は脾臓でFCM測定すること、尻尾DNA等を用いてPCRやCGHアレイや次世代シークエンサー等を用いて網羅的な解析を行い、ヒト21番染色体もしくはその断片の保持領域を解析すること、各種組織(例えば、脳、胸腺、心臓、肺、肝臓、脾臓、腎臓、小腸、精巣など)をFISH(Fluorescence In Situ Hybridization)法により解析し、ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片の保持又は保持率を測定すること、明暗試験、オープンフィールド試験、高架式十字迷路試験などにより行動異常、認知機能異常などを測定すること、MRI、CTなどにより脳や心臓の異常を測定すること、ヒト21番染色体もしくはその断片上の遺伝子発現をRT-PCR法、マイクロアレイ法、次世代シークエンス法により確認することなどを含む。FISH法では、蛍光標識した特異的オリゴヌクレオチドプローブを用いてヒト21番染色体もしくはその断片上の遺伝子とハイブリダイゼーションさせ蛍光顕微鏡を用いて当該染色体を検出する。このような手法によって、選抜されたラットは、ダウン症の表現型をもつ、ヒト21番染色体もしくはその断片を安定的に有し、子孫伝達可能であることを特徴とするダウン症モデルラットであると判定することができる。
具体的な作製手順は、例えば次のようなステップを含むことができる。
(ステップ1)
CHO細胞等のげっ歯類細胞中において、セントロメア領域にloxPを保持する上記定義のヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片上にEGFP遺伝子をCre-loxPシステムを用いて導入し、ヒト21番染色体領域もしくはその断片又は外因性ラット染色体もしくはその断片が保持されているかをPCR法およびFISH法で確認する。
(ステップ2)
上記改変ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片を微小核細胞融合法にてラットES細胞(雄系統)に導入し、ヒト21番染色体もしくはその断片の領域又は外因性ラット染色体もしくはその断片の領域が保持されているかをPCR法およびFISH法で確認する。
(ステップ3)
上記ラットES細胞を胚盤胞期胚に注入し、仮親の子宮に移植することによってキメララットを作製する。
(ステップ4)
上記キメララット雄からの円形精子細胞、精子又は延長精子細胞とラット雌からの卵子を用いて、卵子内円形精子細胞注入法、卵細胞質内精子注入法又は卵子内延長精子細胞注入法により顕微授精を行って子孫伝達ラットを作製し、尻尾のDNA及び細胞を用いて、ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片が保持されているかをPCR法およびFISH法で確認する。
(ステップ5)
交配によりトリソミーラットを繁殖させる。
(ステップ6)
FCM解析により、血液細胞でのGFP陽性率を測定する。
(ステップ7)
血液細胞系譜特異的な各種抗体を用いたフローサイトメトリー(FCM)解析により、血液細胞系譜での蛍光遺伝子発現陽性率を測定する。
(ステップ8)
各組織を採取し、FISH解析によりヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片の保持率を測定する。
(ステップ9)
各組織におけるヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片上の遺伝子発現をRT-PCR法により確認する。
(ステップ10)
各種行動解析、脳や心臓の異常解析等により、正常ラットとの比較を行う。
本発明のダウン症モデルラットの特徴は、以下のとおりである。
上記定義のヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片上の遺伝子類(例えば非特許文献4及び5に記載される遺伝子類)の存在をPCR解析によって確認できる。また、ラットの血液細胞及び各種の組織においてヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片の存在や保持率をFISH解析、又はフローサイトメトリー解析によって確認できる。
ダウン症ラットモデルには、行動解析により不安様行動、記憶障害などの表現型が認められる。
ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片が後代(子孫)にジャームライントランスミッションにより子孫伝達される。ダウン症モデルラットではヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片が極めて安定に保持されている。
[A.1]微小核細胞融合と薬剤耐性クローンの単離
レシピエント細胞にはヒト21番染色体の長腕セントロメア近傍にloxP配列の導入された改変ヒト21番染色体を保持するKazukiら(Gene Therapy, 18:384-393,2011)に記載のDT40(hChr.21-loxP)を用いた。DT40(hChr.21-loxP)を細胞培養皿で培養し、コンフルエントになった時点で20%FBS、1%ニワトリ血清、10-4M 2-メルカプトエタノール、0.05μg/mlコルセミドを添加したRPMI1640培地に交換し、さらに12時間培養してミクロセルを形成させた。培養液を24mlの無血清DMEM培地に置換し、予め100μ/mlのポリL-リジンでコートした遠心用25cm2フラスコ12本(コーニング)に2mlずつ分注し、37℃で30分培養し、細胞をフラスコの底に付着させた。上清を除去し、予め37℃で保温したサイトカラシンB(10μg/ml,シグマ)溶液を遠心用フラスコに満たし、34℃、8000rpm、1時間の遠心を行った。ミクロセルを無血清DMEM培地に懸濁し、8μm、5μm及び3μmフィルターにて精製した。精製後、1700rpmで10分間遠心し、無血清DMEM培地5mlに懸濁した。
[A.2.1]PCR解析
G418耐性株のゲノムDNAを抽出して鋳型として組換え体を選別するため、以下のプライマーを用いてPCRを行い、hChr.21-loxPがCHO細胞に導入できているかを確認した。そのプライマー配列を以下に示す。
#21CEN<1>2L:5'-aaatgcatcaccattctcccagttaccc -3'( 配列番号1)
PGKr1:5'-ggagatgaggaagaggagaaca-3'( 配列番号2)
D21S265-L: 5'-gggtaagaaggtgcttaatgctc-3'(配列番号3)
D21S265-R: 5'-tgaatatgggttctggatgtagtg-3'(配列番号4)
D21S261-L: 5'-gagggggactgggacaagccctttgctggaagaga-3'(配列番号5)
D21S261-R: 5'-acattaggaaaaatcaaaaggtccaattattaagg-3'(配列番号6)
D21S268-L: 5'-caacagagtgagacaggctc-3'(配列番号7)
D21S268-R: 5'-ttccaggaaccactacactg-3'(配列番号8)
D21S266-L: 5'-ggcttggggacattgagtcatcacaatgtagatgt-3'(配列番号9)
D21S266-R: 5'-gaagaaaggcaaatgaagacctgaacatgtaagtt-3'(配列番号10)
D21S1259-L: 5'-gggactgtaataaatattctgttgg-3'(配列番号11)
D21S1259-R: 5'-cactggctctcctgacc-3'(配列番号12)
CBR-L:5'-gatcctcctgaatgcctg-3'(配列番号13)
CBR-R:5'-gtaaatgccctttggacc-3'(配列番号14)
上記で得られたCHO(HPRT-;hChr.21-loxP))のうち6クローンについて松原ら(FISH実験プロトコール、秀潤社(東京、日本国)、1994)に記された方法でhuman cotI DNAをプローブにしたFISH解析を行ったところ6クローン中、3クローンで80%以上の割合でCHO(HPRT-;hChr.21-loxP)がCHO細胞に導入されていることを確認した。
[B.1]トランスフェクション及びHAT耐性クローンの単離
上記で得られたCHO(HPRT-;hChr.21-loxP)-3にリポフェクション法を用いて遺伝子導入を行い、90%コンフルエント状態になった6ウエル(well)の細胞に対して、Cre1μgとKazukiら(Gene Therapy,18:384-393,2011)に記載のEGFP挿入ベクター(I-EGFP-I-loxP-3’HPRT)2μgを市販(インビトロジェン)のプロトコールに従い導入した。HAT選択培養下で2週間培養すると、耐性コロニーが出現し、1回の導入で得た合計6個のコロニーを単離し増殖させ、以後の解析を行った(クローン名:CHO(hChr.21-loxP-EGFP))。
[B.2.1]蛍光顕微鏡観察によるGFP挿入体の確認
クローニングした6個のコロニーを蛍光顕微鏡下にて観察したところ、全てのクローンにてGFP陽性細胞が観察され、その陽性率はほぼ100%であった。
HAT耐性株のゲノムDNAを鋳型として組換え体を選別するため以下のプライマーを用いてPCRを行い、部位特異的にGFP遺伝子の挿入が起こっているかを確認した。そのプライマー配列を以下に示す。
TRANS L1:5'-tggaggccataaacaagaagac-3'(配列番号15)
TRANS R1:5'-ccccttgacccagaaattccA-3'(配列番号16)
上記で得られたCHO(hChr.21-loxP-EGFP))のうち6クローンについて松原ら(FISH実験プロトコール、秀潤社、1994)に記載された方法でhuman cotI DNAをプローブにしたFISH解析を行ったところ、6クローン中、6クローンで80%以上の割合でhChr.21-loxP-EGFPが1コピーCHO細胞に導入されていることを確認した。
hChr.21-loxP-EGFPを保持するキメララットを作製するために、実施例1で得られたhChr.21-loxP-EGFPを保持するCHO細胞からラットES細胞へ微小核細胞融合法により導入した。
[A]hChr.21-loxP-EGFPを保持するキメララットの作製
上記実施例2で得られたhChr.21-loxP-EGFP保持ラットES細胞クローンを用いてHirabayashiらの方法(Mol Reprod Dev.2010 Feb;77(2):94.doi:10.1002/mrd.21123.)でキメララットを作製した。宿主としてはCrlj:WIラット(白色、日本チャールスリバー社(横浜、神奈川、日本国)より購入)の雌雄交配により得られる胚盤胞期胚を用いた。注入胚を仮親に移植した結果生まれる仔ラットは毛色によりキメラであるかどうかを判定できる。hChr.21-loxP-EGFP保持ESクローン(例えばRat ES(hChr.21-loxP-EGFP)2、上記実施例2で取得したもの)を注入した125個の胚を仮親に移植した結果、65匹のキメララット(毛色に濃茶色の部分の認められる)が誕生した。そのうち53匹は毛色が有色と観察できるキメラ個体であった(図3)。すなわち、hChr.21-loxP-EGFPを保持するラットES細胞株はキメラ形成能を保持している、すなわちラット個体の正常組織に分化する能力を保持していることが示された。
上記キメララットから交配により繁殖させようと試みたが、GFP陽性個体得られなかった。次に上記原因はトリソミー個体の雄の不妊が考えられたことから、精細管におけるGFP陽性率を観察した結果、18匹中1匹で陽性個体が観察された(図4、図5)。上記GFP陽性キメラマウス雄(キメラ個体番号#1753-2)(図5上図)のGFP陽性の精細管から円形精子細胞を取り出し、FACSにてGFP陽性画分をソーティングしてから以下の実験に用いた。
[A]実体蛍光顕微鏡観察
上記で得られたTC(hChr.21-loxP-EGFP)ラットについて脳、胸腺、心臓、肺、肝臓、腎臓、脾臓、小腸、骨格筋及び精巣を実体蛍光顕微鏡観察下にて観察したところ、全ての組織にてGFP陽性が観察され、その陽性率は100%であった。その代表的な結果を図8に示す。
末梢血でのGFP陽性率が90%以上であった個体について、血液細胞系譜特異的な各種抗体(バイオレジェンド、ebioscience)を用いたFCM解析により、血液細胞系譜でのGFP陽性率を検討したところ、末梢血、骨髄及び脾臓のいずれにおいてもCD61+/CD45RA-(NK細胞様)、CD3+/CD4+(CD4+T細胞)、CD3+/CD8+(CD8+T細胞)、CD45R+(B細胞様)におけるGFP陽性率は90%以上であった。その代表的な結果を図9に示す。
上記と同様の個体の組織において、Shinoharaらの報告(Human Molecular Genetics,10:1163-1175,2001)に記された方法でHuman CotI DNA(R)(Thermo Fisher Scientific)をプローブにしたFISH解析を行ったところ、視覚的にhChr.21-loxP-EGFPの存在を確認し、84~100%の細胞でhChr.21-loxP-EGFPが存在していることが確かめられた。その代表的な結果を図10に示す。
TC(hChr.21-loxP-EGFP)雌ラットとCrlj:WIラット(白色、日本チャールスリバー社(横浜、神奈川、日本国)より購入)雄を交配することで伝達率を検討した。F2世代からF6世代までで309匹の仔ラットが得られ、181匹がGFP陰性、128匹がGFP陽性個体であった(伝達率は41.4%)。すなわち、伝達率はメンデル遺伝の法則(理論値50%)よりも少し低下しているものの、hChr.21-loxP-EGFP形質が41.4%の頻度で出現したことが確かめられ、卵子におけるhChr.21-loxP-EGFPの保持率が80%以上であることが示された。
TC(hChr.21-loxP-EGFP)ラットの脳、胸腺、心臓、肺、肝臓、腎臓、脾臓、小腸、骨格筋及び精巣において、トータルRNAを市販のプロトコール(QIAGEN)に従い抽出後、市販のプロトコール(Invitrogen)に従い、cDNA合成を行い、それを鋳型としてPCRを行い、ヒト21番染色体上の遺伝子発現を検出した。そのプライマー配列を以下に示す。
ヒト21番染色体上の遺伝子発現検出用プライマー:
APP-L:5'-gccccgtaaaagtgttaca-3'(配列番号17)
APP-R:5'-acgtttgtttcttcgtgcct-3'(配列番号18)
SOD1-1L:5'-attctgtgatctcactctcagg-3'(配列番号19)
SOD1-1R:5'-tcgcgactaacaatcaaagt-3'(配列番号20)
IFNAR2-1L:5'-cgaagtttcagtcggtgag-3'(配列番号21)
IFNAR2-1R:5'-ggcattcaggttttatccc-3'(配列番号22)
TTC3-1L:5'-tggacaaatataaggcatgttca-3'(配列番号23)
TTC3-1R:5'-gtcaccttcctctgcctttg-3'(配列番号24)
ETS2-1L:5'-taccatgccaatggtttataagg-3'(配列番号25)
ETS2-1R:5'-atgtgactgggaacatcttgc-3'(配列番号26)
PCP4-1L:5'-gaattcactcatcgtaacttcattt-3'(配列番号27)
PCP4-1R:5'-ccttgtaggaaggtatagacaatgg-3'(配列番号28)
MX1-1L:5'-tggactgacgacttgagtgc-3'(配列番号29)
MX1-1R:5'-ctcatgtgcatctgagggtg-3'(配列番号30)
TFF3-1L:5'-ggctgtgattgctgccag-3'(配列番号31)
TFF3-1R:5'-gtggagcatgggacctttat-3'(配列番号32)
TFF1-1L:5'-cagggatctgcctgcatc-3'(配列番号33)
TFF1-1R:5'-atcgatctcttttaatttttaggcc-3'(配列番号34)
コントロール遺伝子発現検出用プライマー:
GAPDH-F: 5’-ccatcttccaggagcgaga-3’(配列番号35)
GAPDH-R: 5’-tgtcataccaggaaatgagc-3’(配列番号36)
[A]明暗往来試験
TC(hChr.21-loxP-EGFP)ラット系統を用い、GFP陽性の個体をダウン症モデルラット(以下、「DS」と称する。)、GFP陰性の個体をコントロール(以下、「Control」と称する。)として使用した。10週齢の雄を各グループ7匹ずつ準備した。実験者に触られることによるストレスを減らすため、9週齢の時点でヒトへの馴化(ハンドリング5分)を行った。これらの動物を使用して、RIKENのウェブページ(http://ja.brc.riken.jp/lab/bpmp/SOPs/index_mc.html)に記された方法を参考にして明暗往来試験を実施した。
10週齢のTC(hChr.21-loxP-EGFP)ラットの雄をDS,controlグループそれぞれで7匹ずつ準備した。9週齢の時点でヒトへの馴化(ハンドリング5分)を行い、明暗往来試験終了後1日以上空けてオープンフィールド試験を行った。Kaloudaらの報告(Pharmacology,Biochemistry and Behavior,138:111-116,2015)に記された方法を参考にして、以下のようにオープンフィールド試験を実施した。行動解析部屋に動物を慣れさせるため、飼育部屋から行動解析部屋に動物を移動してから1時間以上経過して実験を開始した。昼光色の電球により装置中央の光量を30ルクスに設定されたラット用角型オープンフィールド(70cm×70cm、室町機械株式会社(東京、日本国))の角から壁に沿って動物を入れ、10分間の動物の行動データをビデオトラッキングソフトウェア(ANY-maze、Stoelting社)により取得した。総移動距離に対する中心滞在時間(中心エリア:30cm×30cm)を解析したところ、DSでその割合が低い傾向にあった(図13)。以上の結果から、DSで不安様行動増加が示された。
10週齢のTC(hChr.21-loxP-EGFP)ラットの雄をDS,controlグループそれぞれで6匹ずつ準備した。9週齢の時点でヒトへの馴化(ハンドリング5分)を行い、明暗往来試験、オープンフィールド試験に使用した動物を高架式十字迷路試験にも使用した。
Claims (9)
- ヒト21番染色体もしくはその断片上の少なくとも1つの遺伝子と相同なラット遺伝子がトリソミーである、かつ、子孫伝達可能であることを特徴とするダウン症モデルラット。
- ヒト21番染色体もしくはその断片、又はヒト21番染色体もしくはその断片と相同なラット遺伝子が存在する外因性ラット染色体もしくはその断片、を含み、前記ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片上の少なくとも1つの遺伝子と、当該遺伝子と相同な内因性ラット遺伝子とが加わりトリソミーである、かつ、子孫伝達可能であることを特徴とするダウン症モデルラット。
- 前記ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片が、子孫ラットの組織内で80~90%以上の保持率で保持されている、請求項2に記載のダウン症モデルラット。
- 正常ラットと比較して、不安様行動又は記憶障害を含む行動異常を有する、請求項1、2又は3に記載のダウン症モデルラット。
- 前記ヒト21番染色体の断片又は外因性ラット染色体の断片が、セントロメア領域を含み、かつ10~34Mbのサイズを有する、請求項2~4のいずれか1項に記載のダウン症モデルラット。
- 前記ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片が、蛍光タンパク質をコードするDNAを含む、請求項2~5のいずれか1項に記載のダウン症モデルラット。
- 請求項2~6のいずれか1項に記載の子孫伝達可能なダウン症モデルラットの作製方法であって、ヒト21番染色体もしくはその断片を含む、又はヒト21番染色体もしくはその断片と相同なラット遺伝子が存在する外因性ラット染色体もしくはその断片を含む、ミクロセルと雄系統のラットES細胞とを微小核細胞融合法により融合し、それによりヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片を含み、前記ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片上の少なくとも1つの遺伝子と、当該遺伝子と相同な内因性ラット遺伝子とが加わりトリソミーであるES細胞を作製する工程、前記ES細胞をラット胚盤胞期胚又は8細胞期胚に導入し、得られた胚をラット仮親の子宮に移植してキメララットを作製する工程、得られた雄キメララットから得た円形精子細胞をラットの卵子に卵子内円形精子注入法(ROSI)により顕微受精し、或いは卵子内延長精子細胞注入法(ELSI)により上記雄キメララットの延長精子細胞とラットの卵子を顕微受精し、或いは卵細胞質内精子注入法(ICSI)により上記雄キメララットの精子とラットの卵子を顕微受精し、子孫ラットを作製する工程、並びに、前記子孫ラットからヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片を保持する子孫伝達可能なラットを選別する工程を含む方法。
- 前記ヒト21番染色体もしくはその断片又は外因性ラット染色体もしくはその断片が、蛍光タンパク質をコードするDNAを含む、請求項7に記載の方法。
- 前記ヒト21番染色体の断片又は外因性ラット染色体の断片が、セントロメア領域を含み、かつ10~34Mbのサイズを有する、請求項7又は8に記載の方法。
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