| Background and ObjectiveLeukemia is the most common children malignancy, accounting for almost30%of children cancer. Although the causes of leukemia are largely unknown, a few prenatal chromosomal and genetic alterations, such as trisomy21(Down syndrome) and TEL-AML1rearrangements have been associated with an increased risk of children leukemia, and studies of twin leukemia have revealed that leukemia can be initiated in utero. Ras are membrane-associated small GTPases and play a critical role in transducing signals, and the mutations at codons12and13cause hyperactive Ras signaling, which leads to constitutive activation of the Ras protein and affect the cell proliferation/differentiation, survival and malignant transformation. Pten (phosphatase and tensin homologue), as a negative regulator of the PI3K-Akt pathway, is involved in cell cycling, lineage commitment, mobilization, and hematopoietic stem/progenitor cell function. Both gain-of-functional Kras mutation(s) and loss of Pten expression have been implicated in children leukemia.Taken together, we raised the hypothesis that both oncogenic K-ras mutation(s) and Pten deficiency could occur in utero as an early event which contributes to postnatal leukemogenesis. Most existing murine models on these two genetic alterations use adult bone marrow cells and are not appropriate to address this hypothesis.Cre transgenic mice can be used to delete gene sequences flanked by loxP sites in specific tissue. In Vav-iCre transgenic mouse, the iCre transgene is expressed under the control of murine Vav gene regulatory elements. The expression of Vav gene is exclusively restricted to hematopoietic cells in adult mice and may be in hematopoietic stem cell (HSC)-derived progenitors in earliest prenatal. Therefore, Vav-iCre-mediated recombination occurred in most hematopoietic cells of all hematopoietic organs and the Vav-iCre transgene mice are effective tools for constructing fetal-stage-blood-lineage specific nonviral-based genetic modified murine models. In LSL-KrasG12D mice, there is a transcriptional termination codon flanked by loxP sites upstream of a mutation of glycine to aspartic acid encoded by codon12in exonl. Excision of the stop cassette by iCre recombinase allows the expression of oncogenic Kras. The conditional Pten konckout mice (Ptenf/f) were inserted two loxP sequences on either side of the exon5, which encode the phosphatase domain and erased by iCre recombinase leading to disruption of Pten.In this study, by using LSL-KrasG12D mice, Ptenf/f mice, and Vav-iCre transgene mice, we sought to constructed two fetal-stage-blood-lineage specific nonviral-based genetic modified murine models, and obtain stable and uniformed overexpression or deletion of the gene of interest, to evaluate the effect(s) of deregulated KrasG12D and Pten during fetal hematopoiesis and their potential roles in postnatal leukemogenesis. This study can be divided into two parts:Part Ⅰ:Effects of oncogenic Kras G12D+in murine postnatal leukemogenesis and fetal hematopoiesisMethods1. Crossed LSL-KrasG12D mice and Vav-iCre mice, extract DNA from ear tissues of mice at21days after birth, identified the genetypes of offsprings by PCR and gene sequencing.2. Set up timed matings in the early evening and check the next morning for the presence of a vaginal plug (0.5days post coitus (E0.5)). Euthanize pregnant mice with carbon dioxide on E14.5and E15.5, carefully dissect and gather the embryos, HE stain the parasagittal section, observe the differences on gross and histological morphology; collect fresh fetal liver cells and check the expressions of CD71, Ter119by flow cytometric analysis, compare the percentage of primitive progenitor cells and proerythroblasts. Results1. Among the first48offsprings, there are17Kraswt;Vav-Cre-mice (35.24%),16LSL-KrasG12D mice (33.33%) and15Kraswt;Vav-Cre+mice (31.25%), but no viable KrasG12D+;Vav-iCre+pups were identified, which is not consistant with the Mendelian inheritance. No KrasG12D mutation was found in15Vav-iCre+littermate mice by sequencing on purified genotyping PCR products.2. Compared with Kraswt;Vav-Cre+embryos, the E14.5KrasG12D+embryos have pale-colored liver and small hemorrhage foci, the E15.5KrasG12D+embryos appear brain or extensive systemic hemorrhage. In hematoxylin and eosin (HE) stained fetal liver parasagittal section from E15.5KrasG12D+embryos, mild decrease in cellularity in the proximal portion of and increased erythroblasts in the distal portion of the KrasG12D+hepatic lobe were observed. Moreover, severe cell death was observed in the distal portion of the KrasG12D+hepatic lobe.3. Cells isolated from E14.5Kraswt;Vav-Cre+or KrasG12D+fetal livers were stained with anti-CD71and anti-Ter119for flow cytometric analysis (FCAS). It displayed an increased Ter119-CD71+populations ((8.20±4.39)%) in KrasG12D+fetal livers compared with (3.66±0.62)%in Kraswt;Vav-Cre+fetal livers. The difference is significant (P<0.001)。Summary1. No live KrasG12D+; Vav-iCre+pups were detected by crossing LSL-KrasG12Dmice and Vav-iCre mice.2. KrasG12D+embryos died of brain or extensive systemic hemorrhage between E14.5to E15.5.3. KrasG12D+embryos fetal livers displayed an increased percentage in primitive progenitor cells and proerythroblasts. Part Ⅱ:Effects of Pten deletion in murine postnatal leukemogenesis and fetal hematopoiesisMethods1. Crossed Ptenf/f mice with Vav-iCre to get Pten+/-mice, then crossed Pten+/-mice with Ptenf/f mice. Identified the genotypes of offsprings, and divided into observation group and control groups. Monitor mice everyday, detecte the complete blood count CBC using peripheral blood (PB) on50day after birth. Euthanize badly sick mice with carbon dioxide, and collect PB to do cytospin and Giemsa stain, fix spleen, liver, thymus tissues and HE stain, isolate bone marrow (BM) cells for FCAS. Meanwhile, dealed with the control groups mice as the same way, and analyse their median survival difference.2. mice BM cells/thymocytes transplantation:4×106BM cells or thymocytes harvested from Pten-/-mice (CD45.2) were injected intravenously via the tail vein into sub-lethally irradiated (650cGy)6to8-week-old CD45.1/CD45.2mice. Mice were monitored everyday, and were euthanized when badly sick. Fresh cells harvested from BM and spleen were subsequently subjected to FCAS utilizing CD45.1, CD45.2and CD4/CD8antibody. The percentage of CD45.1-CD45.2+was calculated as engraftment cells, and a threshold of2.5%CD45.1-CD45.2+cells was established as a reliable preditor of positive engraftment.3. Set up timed matings in the early evening and check the next morning for the presence of a vaginal plug (0.5days post coitus (E0.5)). Euthanize pregnant mice with carbon dioxide on E14.5, carefully dissect and gather the embryos, observe the differences in gross morphology; collect fresh fetal liver cells for FACS, compare the percentage difference of hematopoietic progenitor cells (HPC).Results1. By crossing Ptenf/f mice with Vav-iCre mice, Pten+/-mice occupied50%in offsprings and live normally more than200days. Among the61offsprings by crossing Pten+/-mice with Ptenf/f mice, there are13Pten-/-mice (21.31%),17 Pten+/-mice (27.87%),15Ptenf/f mice (25.00%) and16Ptenf/+mice (26.23%), which is consistant with the Mendelian inheritance. Almost all Pten-/-mice died with a median survival of82days, which was significantly shorter than that of Pten+/-mice(P<0.001).2. CBC showed the white blood cells (WBC,(36.22±12.46)×109) in Pten-/-mice is signicantly higher than that in Pten+/-mice (7.17±2.01)×109, P<0.05).3. Compared with Pten-/-mice, the Pten-/-mice showed hunched posture, respiratory distress, ruffled fur and weight loss (incidence100%). Necrospy displayed the presence of a thymic mass accompanied by hepatosplenomegaly (incidence85.7%).4. Histological examination of the thymus revealed that the normal thymic architecture was effaced and infiltrated by monomorphic lymphoblastic cells with prominent nucleoli and scant cytoplasm. Leukemic cells infiltrated organs such as spleen and liver. Variable leukemic blasts were present in the peripheral blood of diseased mice.5. Tumor thymocytes were examined for their expression of Thyl and CD19, markers for T cells and B cells respectively. Most of these cells were Thyl+but CD19-((80.2±2.7)%). The CD45+populations were analysed using CD4and CD8, and it showed three (1/2) of the six examined tumors consisted of predominantly CD4+CD8+cells, one (1/6) contained primarily CD4-CD8+cells and the other two (1/3) consisted of CD4+CD8-cells.6. Mice BM cells/thymocytes transplantation test showed the recipient mice were sick in2-16week after injection. FACS results showed the percentage of CD45.1-CD45.2+populations in BM of recipient mice are more than5%((24.4±3.1)%), and the populations are CD3+.7. FACS on fetal liver cells revealed that there was no reduction in the hematopoietic stem cell (HSC) enriched lin-sca-1+c-kit+(LSK) population. However, there was an increased percentage of common myeloid progenitor (CMP) population as well as a decreased percentage of common lymphoid progenitor (CLP). Summary1. By crossing Ptenf/f mice with Vav-iCre mice, Pten+/-mice were born and live normally. By crossing Pten+/-mice with Ptenf/f mice, alive Pten-/-mice were born. The median survival of Pten-/-mice was significantly shorter than that of Pten+/-mice.2. Pten-/-mice developed into T-ALL/L ultimately.3. The T-ALL/L occurred in Pten-/-mice was transplantable。4. Pten-/-mice showed an increased percentage of CMP population as well as a decreased percentage of CLP in fetal liver hematopoietic progenitor cells.Conclution1. K-rasG12D mutation introduced by Vav-iCre during development leads to fetal lethality, suggesting that this murine model cannot be used to assess the leukemogenic ability of K-ras in utero.2. Oncogenic KrasG12D+expression during development may block the differention from erythroid progenitor cells to mature erythrocytes, which might be the cause of anemia.3. None of Pten+/-mice with one copy Pten deletion during fetal stage exhibited T-ALL/L, while Pten-null mice lead to transplantable T-ALL/L.4. Pten deletion during fetal stage hardly affect the overall fetal hematopoiesis except a skewed lymphoid/myeloid development at the progenitor level。... |
PDF Full Text Request