The Establishment Of Ptpn11^D61G/+ Transgenic Mouse Model With Fam210b Depletion In Blood Cells And Its Potential Role In Ptpn11^D61G/+ Blood Cells

[Background]Myelodysplastic/myeloproliferative tumors(MDS/MPN)are a rare group of clonal hematopoietic malignancies.These include chronic granulomonocytic leukemia(CMML),atypical chronic myelogenous leukemia(aCML),juvenile granulomonocytic leukemia(JMML),MDS/MPN(MDS/MPN-RS-T)with cyclic iron granulocytes and thrombocytopenia,and unclassified MDS/MPN(MDS/MPN-U).Adolescent granulocyte monocytic leukemia(JMML)is the only childhood disease with germ-line or somatic mutations,constitutive activation of RAS/MAPK signaling pathway,and clinical features of myelodysplasia and cytopenia with monocyte and granulocyte lineages.Hematopoietic stem cell transplantation is currently the standard treatment for most patients.The hypersensitivity of myeloid progenitor cells to cytokine granulocytemacrophage colony-stimulating factor(GM-CSF)and interleukin-3(IL-3)is a key pathological feature of the disease.The most common gene mutation in JML-like MPN is Ptpn11.The Ptpnll gene encodes the non-receptor protein tyrosine phosphatase SHP2,which is widely expressed in the cytoplasm,nucleus and mitochondria.SHP2 contains two Src homologous 2 domains(N-SH2 and C-SH2)arranged in series with N-terminals and a tyrosine phosphatase(PTP)catalytic domain.In the inactive state,aspartic acid at site 61 and tyrosine at site 62 insert into the catalytic gap of the PTP domain and close the active site,maintaining the self-inhibiting state of SHP2 and inhibiting the phosphatase activity.SHP2 is a key component of signal transduction pathways that linked to developmental processes and hematopoiesis,as well as a key regulator of many signal transduction processes and pathways.SHP2 is critical in the regulation of biological responses to growth factors,hormones,cytokines,and cell adhesion molecules;It has also been linked to a variety of developmental disorders and cancer-related diseases.Current studies on SHP2 mainly focus on the discovery and clinical efficacy of inhibitors,especially allosteric inhibitors of SHP2.Our research group’s previous experimental results found that the Ptpn11E76K/+ model mouse developed leukemia,so bone marrow cells of the wild type and mutant model mice were sequenced by RNA,and it was found that the expression of a gene Fam210b was nearly doubled in the mutant mice.Fam210b gene is a newly identified gene,a mitochondrial localized protein,in recent years.In the terminal erythrocyte differentiation stage,FAM210B protein is highly upregulated,which affects the expression of regulation genes related to erythroid differentiation and thus affects erythroid differentiation.Recent studies have shown that FAM210B interact with the subunits of mitochondrial ATP synthetase,such as ATP5A and ATP5B;Fam210b knockout also reduced the level expression of coenzyme Q10,resulting in decreased oxygen consumption and cell metabolism in anaerobic glycolysis.Therefore,FAM210B also affects mitochondrial energy metabolism.Fam210b is also associated with breast cancer occurrence,metastasis,low survival rate and globin transformation of hemoglobin.In conclusion,because the level expression of Fam210b is significantly decreased in Ptpn11E76K/+ mice,we want to continue to explore the effect of Fam210b knockout in blood cells in Ptpn11D61G/+model mouse on the development of myeloproliferative tumors in mice.[Methods]① Mice breeding:Mx1Cre/+;Fam210bF/+ was propagated with Ptpn11D61G/+to obtain Ptpn11D61G/+;Mx1Cre/+;Fam210bF/+.Then the mice were propagated with Fam210bF/F to obtain Ptpn11D61G/+;Mx1Cre/+;Fam210bF/F homozygous mice.The obtained homozygous mice and Fam210bF/F propagated to obtain Ptpn11D61G/+;Mx1 Cre/+;Fam210bF/F,Ptpn11D61G/+;Fam210bF/F,Fam210bF/F.②Gene identification:When the mice were 8-12 days old,the mice genotype was identified by cutting the toes.③ Modeling of mice establishment:The litters of mice at about 8 weeks old were selected and classified by male and female.PIPC drug dosage was calculated according to 0.1ml/g,and the drug was intraperitoneally injected.④ q-PCR:To verify the level expression of Fam210b in bone marrow cells and observe whether the target gene is knocked out.⑤Peripheral blood collection by tail vein:Flow cytometry was used to detect the proportion of blood cells in peripheral blood of model mouse of different ages.⑥Flow cytometry:Cells were extracted from peripheral blood,spleen and bone marrow of mice,and cell proportion and absolute number of these cells were counted.[Results]1.q-PCR confirmed that the expression of Fam210b in bone marrow cells of Ptpn11D61G/+ genotype mice was decreased compared with Ptpn11+/+ mice.2.The Ptpn11D61G/+ model mouse with Fam210b knockout in blood cells was established successfully.3.Flow cytometry results showed that Fam210b depletion in blood cells did not affect the percentage and absolute count of peripheral blood cells in Ptpn11D61G/+mice.4.Fam210b depletion in blood cells did not affect the spleen weight of Ptpn11D61G/+mice,but reduced the percentage of myeloids in the spleen.5.The proportion of long-term hematopoietic stem cells in bone marrow of Ptpn11D61G/+ mice were significantly higher than Ptpn11+/+ mice.Fam210b depletion in blood cells possibly affects the percentage of LT-HSC in the bone marrow of Ptpn11D61G/+mice.[Conclusion]1.The level expression of Fam210b was decreased in bone marrow of Ptpn11D61G/+mice,compared with Ptpn11+/+mice.2.Fam210b inhibited the proportion of myeloid cells in the spleen of Ptpn11D61G/+mice.3.The proportion of long-term hematopoietic stem cells in bone marrow of Ptpn11D61G/+ mice were significantly higher than Ptpn11+/+mice.