Generation and phenotypic characterization of mutant Npm1 allele knock-in mice
| Autor: | Chiou, Ji-Shain, 邱繼賢 |
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| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 100 Acute myelogenous leukemia (AML) is a kind of blood cancers that myeloid hematopoietic cells over proliferation and differentiation immature, it accounts for 40~50 percentage in adult blood disease. Presently, the reason of AML may be related to genetic mutation, in these cases, 50~60 percentage patients of AML will company with Nucleophosmin (NPM) mutation. NPM is one kind phosphorylation proteins rich in nucleolus, in many human hematological malignant tumor, NPM will mutation or translocation with other genes. There NPM mutations in exon 12, its C terminal form nuclear export signal (NES) then cause NPM protein massive accumulation in cytoplasm. The pathogenetic effects of mutated NPM1 protein have been explored by animal models including transgenic or “humanized” knock-in mouse models. The present research’s motivation is to utilize mouse NPMc knock-in technology to manufacture an “canonical” mouse Npm1 mutant knock-in transgenic mouse model without any “humanized” sequence to improve the NPMc gene’s mutation has relative with AML. NPMc mice were generation by the co-laboratory. Brief to describe, a plasmid with NPMc mutant was constructed and make it into embryonic stem cells, then injected the embryonic stem cell carried with NPMc genes into internal space of blastocyst and implant blastocysts into pseudopregnant mother. When transgenic mice were birth and wean, then mice were delivered into FJU animal facilities. NPMc+ genotype of mice was identified with PCR, in the result, we find that the birth litter’s genotyping include normal (NPMwt/wt) or single NPM mutant (NPMwt/c+) mice, but no double NPM mutant (NPMc+/c+) mice. Therefore, retracing to embryonic development time find NPMc+/c+ mice will die before 8.5 day, only heterozygote NPMwt/c+ survival. Further, result of immunohistochemistry conformed that NPMwt/c+ mice’s bone marrow cells have NPM protein accumulation. Three of twenty-four NPMwt/c+ mice developed an age-independent leukocyte proliferation characteristics. The incidence of abnormal leukocyte proliferation in NPMwt/c+ mice is 12.5%. After signs of illness developed, NPMwt/c+ mice developed myeloproliferation in bone marrow, blood, and spleen, but poor B lymphocyte development in bone marrow. illness NPMwt/c+ mice demonstrated a splenomegaly and increase of total number of splenocytes. Flow cytometric analysis has showed the existence of immature myeloid progenitors in blood and spleen. Spleen histochemistry stain has showed blasts infiltration in the spleen. Further to estimate the immune activities of NPMwt/c+ mice’s splenocytes and thymocytes with mitogen stimulation, those T cell abilities for proliferation is decrease, but NO production is upgrade. In addition, a long-term observation on hematologic development of non-illness NPMwt/c+ mice was done. The non-illness NPMwt/c+ mice demonstrated a normal hematologic parameters, except for B-cell and monocyte counts were low. Interesting, results of colony forming unit assay indicates increase in the hematopoietic activities of marrow hematopoietic stem cells (HSC) in NPMwt/c+ mice without sign of illness, but aberration on cobblestone formation while HSCs contacted with stroma microenvironment, suggesting the NPMc mutant may affect the ability of HSC on contact signal expression. In summary, within this canonical NPMwt/c+ mutant knock-in model, mutant NPM protein accumulation in cytoplasm did cause myeloid cells over differentiation and may lead to pathogenesis. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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