Establishing A Inducible Leukemia Mouse Model With ES Cells

Background:Embryonic stem cells are pluripotent cells isolated from the inner cell mass of blastocyst that have the potential for multi-lineage differentiation. Transgenic animals are generated by introducing exogenous gene into ES cells. This approach has been used successfully for the analysis of gene function and regulation. Acute promyelocytic leukemia (APL) is characterized by the t (15;17) translocation which generates the fusion protein promyelocytic leukemia/retinoic acid receptor α (PML/RARα) in nearly all cases. PML/RARα inhibits the differentiation of myeloid cells, which results in promyelocyte accumulation in bone marrow. The PML/RARa protein inhibits PML function and thereby contributes to enhanced cell survival and growth deregulation. It is important to study how PML/RARα causes APL in vivo. However, an inducible animal model for APL is lacking.Objective:This study was to establish a mouse model in which PML/RARa fusion gene can be controlled by the Tet-on system, and to test the impact of the fusion protein on hematopoietic stem and progenitor cells (HSCs/HPCs).Methods:The human PML/RARa (hPML/RARα) was introduced into mouse ES cells via a lentiviral vector. We have confirmed that the lentiviral construct can efficiently deliver hPML/RARa to murine ES cells with normal karyotypes. We have obtained the chimeric mice by injecting ES cells into blastocysts. The expression of hPML/RARa is regulated by dox with the vector containing Tet-on system. The influences of PML/RARα on HSCs/HPCs and the differentiation of hematopoietic cells were examined by CFC, LTC and liquid culture. Moreover, by inducing hPML/RARα expression for a long time with dox in vivo, we detected the development of leukemia by flow cytometry.Results:RT-PCR demonstrated that the expression of hPML/RARa was turned on in ES cells after24hours of dox administration while in bone marrow cells after3days. CFC assay indicated that HPCs were increased in the transgenic mice after10days while myeloid differentiation was enhanced. In addition, the granulocytic differentiation was increased in vitro. Moreover, we found that mice had normal phenotype after the induction for8months in vivo.Conclusions:1. We have successfully established a mouse model, in which hPML/RARa can be conditionally turned on by dox.2. PML/RARa can increase the number of HPCs and the capacity of differentiation to granulocytes.3. PML/RARa transgenic mice have not developed APL with dox administration for8months, indicating either longer induction time is needed or a requirement of second or multiple hit(s) for the development of APL.