Contributions
Abstract: EP583
Type: E-Poster Presentation
Session title: Bone marrow failure syndromes incl. PNH - Biology & Translational Research
Background
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease caused by PIGA mutation of hematopoietic stem cells. Vav is a specific expression promoter of hematopoietic system, and hematopoietic system-specific knockout mice can be obtained by using Vav-iCre.
Aims
This study aims to construct an animal model with a PNH disease phenotype by using Vav-iCre targeted knockout of PIGA gene in the hematopoietic system.
Methods
C57BL/6N ES cells was used for gene targeting. Exon 3-5 was selected as conditional knockout region (CKO region) (Figure 1A). The KO allele was obtained after Vav-iCre-mediated recombination. Vav-iCre mediated deletion of the CKO region resulted in the loss of Piga gene function in mouse hematopoietic stem cells, thus a Piga conditional knockout (CKO) mouse model was constructed. Flow cytometry was used to detect the expressions of Glycosylphosphatidylinositol (GPI) and GPI-anchor protein (GPI-AP) on the surface of erythrocytes, granulocytes, T lymphocytes and B lymphocytes in peripheral blood of normal C57BL/6N mouse and CKO mouse. In addition, hemolysis related indicators (hemoglobin concentration (Hb), lactic dehydrogenase (LDH), total bilirubin (TBIL), indirect bilirubin (IBIL), free hemoglobin (FHb)) and mouse complement levels of C3a, C5a, and C5b-9 of normal C57BL/6N mouse and CKO mouse were measured. Finally, the spleen and bone marrow of two groups of mouse were taken for pathological examination.
Results
The expressions of GPI and GPI-AP in peripheral blood cells of normal C57BL/6N mouse were positive (Figure 1B) and were almost completely absent in CKO mouse (Figure 1C). What’s more, the proportion of the deficiency remained stable from birth. In normal C57BL/6N mouse group, the Hb were (152.7±11.07)g/l, the level of LDH were (439.8±45.11)U/L, the level of TBIL were (1.868±0.646)μmol/l, the level of IBIL were (2.006±0.308)μmol/l,the level of FHb were (168.1±61.00)mg/l; in CKO mouse group, the Hb were (120.6±17.22)g/l, the level of LDH were (832.9±208.9)U/L, the level of TBIL were (3.830±0.493) μmol/l, the level of IBIL were (3.318±0.293) μmol/l,the level of FHb were (354.1±91.03)mg/l. The above hemolysis related indexes were statistically different between the two groups (p＜0.0001，p=0.0034，p=0.0006，p=0.0001，p=0.0029).The level of C5a,C5b-9 in normal C57BL/6N mouse group were (83.80±13.45)ng/ml and (174.3±10.79)ng/l; in CKO mouse group were (116.3±28.45)ng/ml and (217.2±26.39)ng/l, which had statistical difference between two groups(p=0.0495,p=0.0098). The pathological examination showed that the volume of hematopoietic tissue(VOL) in bone marrow of CKO mouse was increased(95%VOL) compared with that of normal mice(90%VOL), and the granulosiderin cells in the spleen of CKO mouse were distributed in small clusters and aggregated in clumps(Figure 1D).
Conclusion
A Piga conditional knock-out mouse model mediated by Vav-iCre was constructed which had stable GPI-deficient and mild hemolysis.
Keyword(s): Mouse model, Paroxysmal nocturnal hemoglobinuria (PNH)
Abstract: EP583
Type: E-Poster Presentation
Session title: Bone marrow failure syndromes incl. PNH - Biology & Translational Research
Background
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease caused by PIGA mutation of hematopoietic stem cells. Vav is a specific expression promoter of hematopoietic system, and hematopoietic system-specific knockout mice can be obtained by using Vav-iCre.
Aims
This study aims to construct an animal model with a PNH disease phenotype by using Vav-iCre targeted knockout of PIGA gene in the hematopoietic system.
Methods
C57BL/6N ES cells was used for gene targeting. Exon 3-5 was selected as conditional knockout region (CKO region) (Figure 1A). The KO allele was obtained after Vav-iCre-mediated recombination. Vav-iCre mediated deletion of the CKO region resulted in the loss of Piga gene function in mouse hematopoietic stem cells, thus a Piga conditional knockout (CKO) mouse model was constructed. Flow cytometry was used to detect the expressions of Glycosylphosphatidylinositol (GPI) and GPI-anchor protein (GPI-AP) on the surface of erythrocytes, granulocytes, T lymphocytes and B lymphocytes in peripheral blood of normal C57BL/6N mouse and CKO mouse. In addition, hemolysis related indicators (hemoglobin concentration (Hb), lactic dehydrogenase (LDH), total bilirubin (TBIL), indirect bilirubin (IBIL), free hemoglobin (FHb)) and mouse complement levels of C3a, C5a, and C5b-9 of normal C57BL/6N mouse and CKO mouse were measured. Finally, the spleen and bone marrow of two groups of mouse were taken for pathological examination.
Results
The expressions of GPI and GPI-AP in peripheral blood cells of normal C57BL/6N mouse were positive (Figure 1B) and were almost completely absent in CKO mouse (Figure 1C). What’s more, the proportion of the deficiency remained stable from birth. In normal C57BL/6N mouse group, the Hb were (152.7±11.07)g/l, the level of LDH were (439.8±45.11)U/L, the level of TBIL were (1.868±0.646)μmol/l, the level of IBIL were (2.006±0.308)μmol/l,the level of FHb were (168.1±61.00)mg/l; in CKO mouse group, the Hb were (120.6±17.22)g/l, the level of LDH were (832.9±208.9)U/L, the level of TBIL were (3.830±0.493) μmol/l, the level of IBIL were (3.318±0.293) μmol/l,the level of FHb were (354.1±91.03)mg/l. The above hemolysis related indexes were statistically different between the two groups (p＜0.0001，p=0.0034，p=0.0006，p=0.0001，p=0.0029).The level of C5a,C5b-9 in normal C57BL/6N mouse group were (83.80±13.45)ng/ml and (174.3±10.79)ng/l; in CKO mouse group were (116.3±28.45)ng/ml and (217.2±26.39)ng/l, which had statistical difference between two groups(p=0.0495,p=0.0098). The pathological examination showed that the volume of hematopoietic tissue(VOL) in bone marrow of CKO mouse was increased(95%VOL) compared with that of normal mice(90%VOL), and the granulosiderin cells in the spleen of CKO mouse were distributed in small clusters and aggregated in clumps(Figure 1D).
Conclusion
A Piga conditional knock-out mouse model mediated by Vav-iCre was constructed which had stable GPI-deficient and mild hemolysis.
Keyword(s): Mouse model, Paroxysmal nocturnal hemoglobinuria (PNH)