Effect of PIKfyve gene deletion on distribution and number of mature immune cells in mice
Objective To establish a mouse model with an inducible deletion of PIKfyve genes throughout the body and hematopoietic system and explore the impact of PIKfyve on immune homeostasis.Methods LoxP sites were inserted at both ends of exon 6 of the PIKfyve gene to establish PIKfyve+/flox mice.PIKfyve+/flox mice were cross-bred to produce PIKfyveflox/flox mice.PIKfyveflox/flox mice were cross-bred with UBC-Cre/ERT2 mice to obtain PIKfyve+/flox,UBC-Cre/ERT2 mice and PIKfyveflox/flox,UBC-Cre/ERT2 mice,the target mice.Genotyping was performed using tail gene PCR,and deletion efficiency was identified post-induction via real-time quantitative PCR(qPCR)and Western blotting.The proportion and number of mature immune cells in peripheral blood,bone marrow,and spleens were observed and compared between the knockout mice and control mice.A similar breeding strategy was adopted to introduce PIKfyveflox/flox,MX1-Cre mice to confirm the aforementioned results in a distinct mouse model.Results Successful generation of PIKfyveflox/flox,UBC-Cre/ERT2 mice was confirmed based on mouse breeding and tail gene PCR.After induction,a significant decrease in PIKfyve mRNA and protein contents in the knockout mice indicated the establishment of a proper model.Peripheral blood analysis revealed a significant reduction in white blood cells,lymphocytes,neutrophils,and monocytes in the PIKfyve-deficient mice.Flow cytometry demonstrated an overall decrease in the proportions of B cells and NK cells,with a significant increase in T cells and neutrophils,while the overall number trended down.Results from PIKfyveflox/flox,MX1-Cre mice were consistent with those of PIKfyveflox/flox,UBC-Cre/ERT2 mice.Conclusion A workable PIKfyve knockout mouse model has been established,suggesting that PIKfyve can regulate the number and proportions of mature immune cells while making a difference to immune homeostasis.
PIKfyvegene knockoutmouse modelimmune cellshematopoietic system
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