JP5403536B2 - Transgenic non-human animal introduced with human MLL / AF4 fusion gene - Google Patents

Description

  The present invention relates to a transgenic non-human animal into which a human MLL / AF4 fusion gene has been introduced.

  The MLL gene (Mixed lineage leukemia) produces a fusion gene by chromosomal translocation frequently seen in various acute leukemias.

  In particular, the frequency of 11q23 translocation is about 5 to 10% for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) as a whole. It is known to appear as high as 80% and 50-60% of AML. The 11q23 translocation has also been observed in treatment-related (secondary) leukemia (TRL) induced by drug treatments such as topoisomerase II inhibitors.

  Based on recent molecular biological analysis results, there are at least three types of translocation-related genes (PLZF, MLL, RCK) in the 11q23 region, of which “MLL” is involved in the development of infant leukemia and TRL. It has become clear that there is. In addition, more than 20 genes, such as 4q21 (AF-4 / FEL / LTG4), 6q27 (AF-6), 9p22 (AF-9 / LTG9), 19p13 (ENL / LTG19), have already been identified as partner genes in reciprocal translocations. ing. It is thought to cause different diseases depending on the binding partner.

  Of these, MLL / AF8 has already been cloned for a short gene called AF8. In addition, for MLL / AF4 of the present invention, MLL / AF4 fusion gene-introduced mice in which MLL is derived from a mouse and AF4 is derived from a human have been prepared (see Non-Patent Documents 1 and 2). MLL / AF4 fusion gene-introduced mice that are both human and AF4 have not yet been made.

M Metzler et al. Oncogene (2006) 25, 3093-3103 Weili Chen et al., Blood, 15 July 2006, Vol. 108, Number 2, 660-677

  An object of the present invention is to provide a model animal of hematopoietic tumor such as human leukemia and lymphoma.

  The inventor of the present application divides a 7-KB human MLL / AF4 fusion gene into seven, clones it separately by RT-PCR method, integrates them into three, and then integrates them into one to obtain the full-length sequence of MLL / AF4 cDNA. Cloning was successful. Thereafter, in order to produce a transgenic mouse, MLL / AF4 cDNA was incorporated into a virus vector for producing a transgenic mouse, and a set of DNA fragments expressing MLL / AF4 cDNA was excised with an appropriate restriction enzyme. The MLL / AF4 cDNA expression set DNA fragment was injected into a fertilized egg into a mouse fertilized egg, and a transgenic mouse incorporating this gene was successfully produced. Out of the 40 types of mice that were created, the selection of mice that were more likely to give birth to children from the 3 types that were able to maintain the strains resulted in the establishment of strain maintenance. The created transgenic mice were confirmed to develop bone marrow failure symptoms and leukemia and lymphoma.

That is, the present invention is as follows.
[1] A transgenic non-human animal into which a human MLL / AF4 fusion gene has been introduced.
[2] The transgenic non-human animal of [1], wherein the human MLL / AF4 fusion gene consists of the base sequence represented by SEQ ID NO: 1.
[3] The transgenic non-human animal according to [1] or [2], which has a human MLL / AF4 fusion gene on a chromosome and expresses human MLL / AF4 protein in somatic cells.

[4] Any of [1] to [3] having the following characteristics (i) to (iii), the following characteristics (iv) and (v), or the following characteristics (i) to (v): A transgenic non-human animal.
(i) Having bone marrow hematopoiesis
(ii) presenting anemia
(iii) Have thrombocytopenia
(iv) Infiltration of B and T lymphocytes in liver, kidney and lung
(v) Presenting with B and T mixed lymphoproliferative disorder

[5] The transgenic non-human animal according to any one of [1] to [4], which can be used as a model animal for tumor or bone marrow failure in humans.
[6] The transgenic non-human animal according to any one of [1] to [5], wherein the non-human animal is a mouse.
[7] Non-human animals are mice, fertilized egg is deposited as consignment number NITE P -688, transgenic non-human animal of [6].

[8] A human MLL / AF4 fusion gene-introduced transgenic mouse comprising the nucleotide sequence represented by SEQ ID NO: 1.
[9] A human MLL / AF4 fusion gene-introduced transgenic mouse vector into which a human MLL / AF4 fusion gene comprising the base sequence represented by SEQ ID NO: 1 has been introduced.

  A fusion gene-introduced mouse in which both MLL and AF4 are derived from humans is a completely new disease model mouse that has never been produced.

  In addition, even if the diseased mouse has the same medical condition, it has a different onset time from the mouse and human-derived MLL / AF4 fusion gene-introduced mice, and develops bone marrow failure symptoms and leukemia and lymphoma. It can be used for analyzing the mechanism of leukemia, and can be used for investigating the efficacy of compounds against leukemia.

Hereinafter, the present invention will be described in detail.
The transgenic non-human animal of the present invention contains a gene encoding a human MLL / AF4 chimeric protein (human MLL / AF4 fusion gene). Here, the gene encoding the human MLL / AF4 chimeric protein encodes the MLL (mixed lineage leukemia) gene and AF4 protein found in the 11q23 translocation chromosome abnormality found in leukemia such as acute lymphocytic leukemia and infant leukemia. The fused gene is fused by translocation (t (4; 11) (q21; q23) translocation). The MLL gene is composed of 36 exons and encodes a protein consisting of 3969 amino acids. The total length of the cDNA is about 15 kb.

  Non-human animals include non-human primates, rodents such as mice and rats, and mammals such as sheep, dogs, cats, horses, cows and the like. A rodent is preferable, and a mouse is more preferable. In the following description, an example using a mouse is given, but a person skilled in the art can also create a knock-in non-human animal into which a human MLL / AF4 fusion gene has been introduced for other animals.

  A gene encoding a human MLL / AF4 chimeric protein can be obtained from a cell in which an MLL / AF4 fusion gene has been generated by translocation. Such cells can be obtained from patients with leukemia such as acute lymphocytic leukemia. A cell line may be used as the cell. For example, total RNA may be extracted from leukemia cells, cDNA may be prepared using reverse transcriptase, and MLL / AF4 fusion cDNA may be amplified using primers. At this time, the restriction enzyme site in the MLL / AF4 fusion gene may be used to amplify each part and then ligated to obtain full-length MLL / AF4 cDNA. The base sequence of the human MLL / AF4 fusion gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the DNA comprising the base sequence is shown in SEQ ID NO: 2.

The human MLL / AF4 fusion gene to be introduced into the non-human animal of the present invention may have a mutation in a part of its sequence. Such a mutation may be SNP (single base substitution) and is at least 80%, preferably 90%, more preferably 95% or more identical to the base sequence of the MLL / AF4 fusion gene represented by SEQ ID NO: 1. DNA, more preferably at least 98% identity. The identity values are the same as those known to those skilled in the art such as BLAST [J. Mol. Biol., 2 15, 403-410 (1990)], FASTA [Methods. Enzymol., 183, 63-98 (1990)]. A numerical value calculated using a sex search program, preferably a numerical value calculated using default (initial setting) parameters in BLAST, or a numerical value calculated using default (initial setting) parameters in FASTA is there.

  Furthermore, the mutant gene of the human MLL / AF4 fusion gene is a mutant gene in which one to several bases are substituted, deleted or added in the base sequence of the human MLL / AF4 fusion gene represented by SEQ ID NO: 1, for example. Yes, or a mutant gene that can hybridize under stringent conditions with a base sequence complementary to the base sequence of the human MLL / AF4 fusion gene represented by SEQ ID NO: 1. Here, the stringent condition is, for example, a condition of “1XSSC, 0.1% SDS, 37 ° C.”, and a stricter condition is a condition of “0.5XSSC, 0.1% SDS, 42 ° C.” More severe conditions are about "0.2XSSC, 0.1% SDS, 65 ° C".

  The mutant of the human MLL / AF4 fusion gene has the same function as the human MLL / AF4 fusion gene represented by SEQ ID NO: 1. That is, when a mutant is introduced into a non-human animal such as a mouse, the non-human animal has (i) bone marrow hematopoietic failure, (ii) anemia, (iii) thrombocytopenia, (iv) liver, Symptoms such as B and T lymphocyte infiltration in the kidney and lung, (v) B and T mixed lymphocyte proliferative disease, etc. are caused.

  The cDNA introduced into the mouse is linked to a promoter that can be expressed in animal cells. Examples of promoters that can be expressed include promoters derived from mammalian cells such as mouse phosphoglycerate kinase (mouse PGK) promoter, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter, retrovirus promoter, polyoma Examples include viral promoters such as viral promoters and adenovirus promoters. In addition, an enhancer that enhances gene expression may be incorporated. The MLL / AF4 fusion gene is operably linked to a promoter and introduced into a vector. Any vector can be used as long as it can induce the expression of the transgene in the body of an animal, and a vector in which a promoter has been previously incorporated may be used. For example, pMSCVpuro, pMSCVneo, and pMSCVhyg (all available from BD Biosciences Clontech) can be mentioned.

  Transgenic mice can be produced, for example, by the method described in Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980). Although the mouse to be used is not limited, for example, C57Bl / 6N mouse (B6 mouse), BALB / c mouse and the like are used.

  The vector into which the above MLL / AF4 fusion gene has been introduced is introduced into a mouse totipotent cell, and the cell is developed into an individual. Examples of totipotent cells include fertilized eggs and early embryos as well as pluripotent cells such as ES cells (embryonic stem cells). When an early embryo is used, an embryo before the 8-cell stage is preferred. The method for introducing the vector into the cell is not limited, and can be performed by the calcium phosphate method, the electric pulse method, the lipofection method, the aggregation method, the microinjection method, the particle gun method, the DEAE-dextran method, or the like. Among these, microinjection into the male pronucleus of a fertilized mouse egg is preferable.

  Next, the cells into which the MLL / AF4 fusion gene has been introduced are transplanted into the oviduct of a recipient mouse. As the recipient mouse, a female that has been mated with a male whose vas deferens has been cut into a pseudopregnant state is preferably used.

  By selecting individuals from which the transgene has been incorporated into somatic cells and germ cells, transgenic chimera mice in which the target MLL / AF4 fusion gene has been knocked in can be obtained. After confirming that the MLL / AF4 fusion gene has been introduced into the transgenic chimeric mouse, the chimeric mouse is mated with a normal mouse to obtain an F1 mouse. Heterozygotes can be selected from so-called F1 animals obtained by crossing a selected germline chimera with the wild type of the same species. The selection of the heterozygote can be assayed, for example, by screening chromosomal DNA separated and extracted from the tail of the F1 animal by Southern hybridization or PCR.

  The homozygote of the knock-in non-human animal of the present invention can be obtained by crossing these heterozygotes.

The transgenic animals of the present invention develop bone marrow failure symptoms and develop leukemia and lymphoma. Moreover, in normal mice, regeneration of the reproducible liver does not occur, bone marrow density decreases, and lymphoma increases. In detail, it has the following characteristics.
(i) Having bone marrow hematopoiesis
(ii) presenting anemia
(iii) Have thrombocytopenia
(iv) Infiltration of B and T lymphocytes in liver, kidney and lung
(v) Presenting with B and T mixed lymphoproliferative disorder

  Moreover, the onset time of said symptom differs with the transgenic mouse which introduce | transduced the fusion gene of mouse | mouth MLL and human AF4. In other words, it takes 3 to 4 months from the time of birth in mice introduced with a fusion gene of mouse MLL and human AF4 (M Metzler et al. Oncogene (2006) 25, 3093-3103; Weili Chen et al. al., Blood, 15 July 2006, Vol. 108, Number 2, 660-677), the human MLL / AF4 fusion gene-introduced mouse of the present invention has symptoms about 6 months after birth. In particular, in the first year of life, many lymphomas are observed in a lung-like manner.

  Furthermore, the transgenic mouse of the present invention has a decrease in leukocytes, which means that the transgenic mouse of the present invention develops leukemia or lymphoma.

As a transgenic mouse of the present invention, for example, a fertilized egg was incorporated on December 11, 2008 by the National Institute of Technology and Evaluation (NITE) Patent Microorganism Depositary Center (NPMD) (Kazusa Kama feet 2- 5-8) mice that have been deposited with consignment number NITE P -688 (MLL / AF4 08/2/8 n = 12) are listed.

  The transgenic mouse introduced with the human MLL / AF4 fusion gene of the present invention can be used as a mouse model for pathological conditions of hematopoietic tumors such as human leukemia and lymphoma involving the MLL / AF4 fusion gene. For example, it can be used for elucidation of the onset mechanism or pathology of leukemia and lymphoma, elucidation of the pathology of bone marrow insufficiency, screening for therapeutic agents, anticancer agents, and blood-enhancing agents for human leukemia. Further, the transgenic mouse of the present invention can be used for molecular biological elucidation of a gene causing leukemia in cooperation with an MLL / AF4 fusion gene, and further, a target molecular drug targeting the MLL / AF4 fusion gene Can be used for screening and development.

For example, in the screening, a test substance that is a therapeutic drug candidate compound is administered to the transgenic mouse of the present invention, and a therapeutic drug for human leukemia is used as an indicator whether or not at least one of the following effects is observed in the transgenic mouse: A compound that can be used as an anticancer agent or a blood-thickening agent can be selected.
(i) The effect of alleviating bone marrow hematopoiesis
(ii) Alleviating anemia
(iii) Effect of increasing platelets
(iv) The effect of alleviating infiltration of B and T lymphocytes into the liver, kidney and lung
(v) Alleviating B and T mixed lymphoproliferative diseases

The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
Cloning of MLL / AF4 cDNA FIG. 1 shows a cloning protocol. As shown in Fig. 1, MLL / AF4 cDNA is divided into seven parts (1) to (7), cloned separately by RT-PCR, and once integrated into three parts (8) to (10). It was further integrated into one (11), and finally cloned as full-length MLL / AF4 cDNA.

  First, RNA was extracted from MLL / AF4 mRNA-expressing human leukemia cells derived from patients with acute lymphoblastic leukemia ALL, and cDNA was prepared using reverse transcriptase. The patient had t (4; 11) (q21; q23), was positive for MLL / AF4, and had an N-ras codon 12 mutation (GGT → GAT (G12D)). CD19, CD34, HLA-DR and CD15 were positive. Thereafter, PCR was performed with Ex Taq polymerase. However, the EcoRI site of (5) and (6) was prepared by causing a base mutation without an amino acid sequence mutation during PCR. Thereafter, (8)-(10) sites were amplified with platinum pfx polymerase. Thereafter, cDNAs corresponding to (3) and (4) were introduced into EcoRI / HindIII / KpnI of the pBluescript vector. Separately, cDNAs corresponding to (3) and (4) were introduced into KpnI / HindIII / BamHI of the pBluescript vector. Then, cDNAs corresponding to (5), (6) and (7) were introduced into BamHI / EcoRI / HindIII / ClaI of pBluescript. However, because (7) has an EcoRI site, (7) was introduced last. (8) (9) (10) was introduced into the EcoRI / KpnI / BamHI / SalI sites of the pUC18 vector. However, since there is an EcoRI site in (10), attention was paid to the order of introduction. Finally, the DNA fragment was excised by cutting with EcoRV / XhoI. pcDNA3.1 (+) was cleaved with EcoRV / XhoI, and the excised DNA fragment (MLL / AF4 cDNA) was introduced. Thereafter, MLL / AF4 cDNA was inserted into EcoRI at the MSC site of the pMSCVneo vector and ClaI upstream of the 3'LTR.

The results of agarose gel electrophoresis analysis of the isolated human MLL / AF4 cDNA are shown in FIG.
The nucleotide sequence of the isolated human MLL / AF4 fusion gene cDNA is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the DNA comprising the nucleotide sequence is shown in SEQ ID NO: 2.

Generation of Transgenic Mice Transgenic mice were generated by the method of Gordon et al .: 1980. Proc. Natl. Acad. Sci. 77: 7380-7384.

  FIG. 3 shows the structure of the MSCVneoMLL / AF4 construction. The cloned MLL / AF4 cDNA was fragmented with Blunt Psp1406I and Nde I restriction enzymes at the EcoR I restriction enzyme site of the pMSCVneo vector. After agarose gel electrophoresis, a 12.6 kb size DNA band was excised by ethidium bromide staining to prepare the intended DNA fragment for injection. The excised DNA was purified using QIAquick Gel Extraction Kit (QIAGEN) (FIG. 4). For microinjection, 200 pronuclear embryos derived from C57BL / 6N Crj mice were used. An injected embryo into which a DNA fragment expressing MLL / AF4 cDNA was injected was transplanted into a recipient mouse (Crj: CD-1, pseudo-pregnant female with plug confirmation) fallopian tube. The baby was born naturally. After weaning at 4 weeks of age, tails were collected and confirmed by gene analysis (PCR method) (FIG. 5). As shown in FIG. 5, pups were further born from the transgenic Founder mouse, and gene transfer was also good in the F1 mouse. Gene expression (RT-PCR method) in each organ was confirmed by Founder and F1 (FIG. 6). As shown in FIG. 6, there was a difference in the expression level of MLL / AF4 cDNA depending on the organ.

The fertilized eggs of the resulting transgenic mice were transferred to the National Institute of Technology and Evaluation (NITE) Patent Microorganism Deposit Center (NPMD) on December 11, 2008 (2-5 Kazusa Kamashika, Kisarazu, Chiba, Japan). -8) to have been deposited in the consignment number NITE P -688. The identification display is MLL / AF4 08/2/8 n = 12.

Analysis of transgenic mouse phenotype Hematological analysis of peripheral blood was performed as needed, and pathological dissection, spleen and bone marrow surface marker analysis were performed by euthanizing surviving mice.

  Peripheral blood was collected from 2-month and 10-month-old transgenic mice (Tg mice) and normal mice (wild-type mice) for hematological analysis. In addition, blood analysis of founder mice (Found.) At 14 and 18 months was also performed. Table 1 shows the results. In the table, WBC is the white blood cell count, RBC is the red blood cell count, Hb is the hemoglobin concentration, and Plt is the platelet count. As shown in Table 1, a decrease in white blood cell count and anemia were observed gradually after birth.

  FIG. 7 shows an excised specimen of the spleen. It is larger than the wild type. Transgenic mice were dissected, spleen, liver, lung and bone marrow were collected, sectioned specimens were prepared, and microscopic observation was performed.

  FIG. 8 shows a weakly enlarged image of the spleen. 8A and B are spleens of transgenic mice. As shown in the figure, in the transgenic mouse, the normal structure of the spleen was destroyed by lymphoma-like cell infiltration.

  FIG. 9 shows the lungs of the transgenic mouse. As shown in the figure, multiple lymphoma infiltration was observed.

  FIG. 10 shows immunostaining of the lungs of the transgenic mouse. As shown in the figure, infiltration of lymphoma cells exhibiting surface trait B and T cell trait was observed in the lung, and lymphoma cell infiltration was also observed in the kidney (FIG. 11).

Many infiltrations of lymphoma cells were observed in the liver tissue. (FIG. 12).
Furthermore, lymphoma cells with clear nucleolus that appear in peripheral blood also appear (FIG. 13). From this result, it was found that this transgenic mouse developed lymphoproliferative disease.

  Furthermore, the bone marrow of transgenic mice and normal mice was observed (FIG. 14). In the transgenic mice, the density of bone marrow hematopoietic cells such as granulocytes and megakaryocytes was relatively low as compared with normal mice, and nucleated lymphocytes were observed to increase in the gaps.

It is a figure which shows the protocol of the cloning of a human MLL / AF4 fusion gene. It is an agarose electrophoresis photograph after EcoRV and XhoI restriction enzyme cleavage of the cloned human MLL / AF4 cDNA. It is a figure which shows the structure of the MSCVneoMLL / AF4 cDNA construction used for introduce | transducing into a mouse | mouth. It is a photograph showing the result of electrophoresis of 12.6 Kb MSCVneoMLL / AF4 cDNA (lane 3) cleaved with Psp1406I and NdeI restriction enzymes, extracted and purified. It is a figure which shows the result of the MLL / AF4cDNA transmission analysis by the DNA of the offspring (F1) tail born from MSCVneoMLL / AF4cDNA expression positive Founder mouse. It is a figure which shows the result of the expression analysis of MLL AF4 cDNA in each organ of the obtained transgenic mouse (F1). It is a photograph of the isolated spleen. (A) shows the spleen of a P230bcr / abl transgenic mouse showing splenomegaly, (B) shows the spleen of an allogeneic normal mouse, and (C) shows the spleen of a transgenic mouse positive for MLL / AF4 cDNA expression. Shows splenomegaly. It is a photograph which shows the weak enlarged image (A) and the strong enlarged image (B) of the spleen of a MLL / AF4 cDNA expression positive transgenic mouse. The normal structure of the spleen is destroyed by lymphoma cell infiltration. It is a photograph of the lung of a transgenic mouse. (A) shows a weakly magnified image and (B) shows a strongly magnified image. Arrow indicates lymphoma cell infiltration. It is a photograph showing the result of immunostaining of lung infiltrating cells. Double staining of CD3 (DAB / brown) and CD45 (FITC / fluorescent green) was performed. The photo is x40x and the top right is x800x. CD3 shows the surface phenotype of T lymphocytes and CD45 shows the B lymphocytes. It is a photograph of the kidney of a transgenic mouse. Arrow indicates lymphoma infiltration. It is a photograph of the liver of a transgenic mouse. (A) shows a weakly magnified image and (B) shows a strongly magnified image. Arrow indicates lymphoma infiltration. It is a photograph which shows a peripheral blood smear. Lymphocyte cells with clear nucleolus are observed. It is a photograph of the bone marrow of a transgenic mouse. (A) shows transgenic mouse bone marrow, and (B) shows normal mouse bone marrow. Transgenic mouse bone marrow reveals infiltration of lymphoma cells with dense nuclei.

SEQ ID NOs: 1 and 2: synthesis

Claims (8)

A transgenic non-human animal into which a human MLL / AF4 fusion gene comprising the base sequence represented by SEQ ID NO: 1 has been introduced. The transgenic non-human animal according to claim 1, which has a human MLL / AF4 fusion gene on a chromosome and expresses human MLL / AF4 protein in somatic cells. The transgenic non-human according to claim 1 or 2 , which has the following characteristics (i) to (iii), the following characteristics (iv) and (v), or the following characteristics (i) to (v): animal.
(i) Having bone marrow hematopoiesis
(ii) presenting anemia
(iii) Have thrombocytopenia
(iv) Infiltration of B and T lymphocytes in liver, kidney and lung
(v) Presenting with B and T mixed lymphoproliferative disorder The transgenic non-human animal according to any one of claims 1 to 3 , which can be used as a model animal for tumor or bone marrow failure in humans. The transgenic non-human animal according to any one of claims 1 to 4 , wherein the non-human animal is a mouse. The transgenic non-human animal according to claim 5 , wherein the non-human animal is a mouse and is produced from a fertilized egg deposited under accession number NITE P- 688.   A human MLL / AF4 fusion gene-introduced transgenic mouse comprising the nucleotide sequence represented by SEQ ID NO: 1.   A human MLL / AF4 fusion gene-introduced transgenic mouse vector into which a human MLL / AF4 fusion gene comprising the nucleotide sequence represented by SEQ ID NO: 1 has been introduced.

Images