Study On Effects Of Vav-iCre Promoter-mediated Knockout Of Autophagy Related Gene On Hematopoietic System In Mice

Objective:Molecular basis for fetal hematopoiesis has not been fully understood.Beclin 1 has been primarily considered as a critical cytoplasmic protein in autophagy pathway,which has a broader physiological role in development and aging,as well as in protecting against pathogen invasion,cancer and certain neurodegenerative diseases.To explore the role of Beclin 1 in hematopoiesis,we generated conditional knockout mice by deletion of biallelic beclin1 in hematopoietic cells.Unlike autophagy defective mice with atg7 deletion surviving through embryonic and neonatal development,Beclin 1deficient fetal mice experienced complete perinatal lethality due to hematopoiesis failure.The bone marrow of the mutant pups exhibited severe deficiency and disrupted quiescence in HSCs.The Colony-Forming Units assay showed that the hematopoietic reconstitution capability of bone marrow HSCs with deletion of biallelic beclin1 were severely impaired.Abnormality was also seen in the red blood cells and platelets in the homozygous deletion mice.These results indicate an essential role of Beclin 1 in embryonic hematopoiesis.Methods:?1?Constructing a mouse model with specific deletion of biallelic beclin1 in the hematopoietic system..The expression of the Cre enzyme was initiated using the Vav promoter which is specifically expressed in the hematopoietic system.To achieve the purpose of deleting the biallelic beclin1 in hematopoietic system,we deleted the sequence between the fourth exon and the seventh exon of the beclin1 gene,resulting in a frame shift mutation.?2?PCR was used to amplify the genomic DNA extracted from mice tails,and genotypes were identified by agarose gel electrophoresis.?3?beclin1^f/f mice were mated with beclin1+/f;Vav-iCre mice?This article is abbreviated as beclin1+/-?,pregnant mice were anesthetized and decapitated,and fetal mice at 18.5 days were removed by caesarean section for relevant experiments.?4?The pathological changes of fetal mice liver were detected by hamatoxylin and eosin staining?HE?.After the fetal liver was removed and placed in 4% PFA for fixation or the fetus was fixed in 4% PFA,the fetal liver was examined.?5?The fetal mice at 18.5,various blood cell indicators of peripheral blood were detected by a three-class blood analyzer.?6?The number and percentage of hematopoietic stem/progenitor cells of mice bone marrow at embryonic 18.5 days was detected by multi-color flow analyzer.?7?The ability of colony formation of long-term hematopoietic stem cells and mononuclear cells of mice bone marrow at embryonic 18.5 days was detected by Colony-Forming Units assay.Results:?1?We first successfully constructed beclin1^f/f mice and beclin1+/f;Vav-iCre mice.The two kind mice were crossed and the offspring had a genotype of beclin1^f/f;Vav-iCre?This article is abbreviated as beclin1-/-?.This offspring was the mice with deleted bialleilic beclin1 in the hematopoietic system.Since the homozygous deletion mice were all dead in the perinatal period,we selected the mice at embryonic 18.5 days for subsequent functional testing.?2?In beclin1^f/f;Vav-iCre fetal mice genomic DNA,the deletion band can be detected by genotyping,indicating that beclin1 was successfully deleted.All mice without the Vav promoter,wild type mice?This article referred to WT?,had no the deletion band.?3?The beclin1^f/f;Vav-iCre mice at embryonic 18.5 days displayed small and pale body.?4?In the beclin1^f/f;Vav-iCre mice liver at embryonic 18.5 days,the red blood cells were decreased in blood vessels;sinusoids were decreased,the number of nucleated cells was increased,indicating an abnormal extramedullary hematopoiesis.The liver begins to develop ischemic necrosis at embryonic 15.5 days,and appears as a white plaque on the appearance of the liver.?5?In the peripheral blood of beclin1^f/f;Vav-iCre mice at embryonic 18.5 days,thewhite blood cells and lymphocyte cells increased,the red blood cells decreased significantly,and the platelet cells increased but the platelet function was abnormal.?6?The proportion of hematopoietic stem and progenitor cells in the bone marrow of beclin1^f/f;Vav-iCre mice at embryonic 18.5 days was reduced significantly.?7?Colony-Forming Units assay showed that both long-term hematopoietic stem cell and mononuclear cells in beclin1^f/f;Vav-iCre mice bone marrow at embryonic 18.5days were lose the ability of colony formation and the bone marrow hematopoietic reconstitution ability was seriously impeded.Conclusion:The biallelic beclin1 deletion mice were dead in the perinatal period.The hematopoietic stem and progenitor cell populations were reduced in the homozygous beclin1 deleted mice.The hematopoietic reconstitution capability of hemopoietic stem cells was impaired severely,and the red blood cells was reduced significantly,the platelet function was abnormal in the homozygous knockout mice.These results indicate an important role of Beclin 1 in embryonic hematopoiesis.