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Abstract
One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem (ES) cells, which is used to produce gene-targeted mice for wide applications in biomedicine. However, a major bottleneck in this approach is that the robustness of germline transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing, which impairs the efficiency and robustness of mouse model generation. Recently, we have established a new type of pluripotent cells termed extended pluripotent stem (EPS) cells, which have superior developmental potency and robust germline competence compared to conventional mouse ES cells. In this study, we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage. Based on gene targeting in mouse EPS cells, we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation, which could be accomplished in approximately 2 months. Importantly, using this approach, we successfully constructed mouse models in which the human interleukin 3 (IL3) or interleukin 6 (IL6) gene was knocked into its corresponding locus in the mouse genome. Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting, which have great application potential in biomedical research.
Highlights
Modified mouse models are invaluable tools in biomedicine because they serve as powerful tools to study gene function, development and human diseases (Visigalli et al, 2010; Carido et al, 2014; Kenney et al, 2016)
We established an extended pluripotent stem (EPS) cell-based approach for efficiently generating mouse models with precise genetic modifications. This approach combines EPS cells, tetraploid complementation and CRISPR/Cas9 technology, which permits the production of mouse models that are precisely genetically modified within 2 months
We successfully generated mouse models that replaced the mouse Il3 and Il6 genes with their human counterparts. These results demonstrate the feasibility of using our approach to efficiently and rapidly create mouse models, especially those that are difficult to produce using conventional gene-targeting strategies
Summary
Modified mouse models are invaluable tools in biomedicine because they serve as powerful tools to study gene function, development and human diseases (Visigalli et al, 2010; Carido et al, 2014; Kenney et al, 2016). Longterm culture of ES cells in 2i medium (Ying et al, 2008), a well-established condition for maintaining ES self-renewal, cannot maintain the genetic and epigenetic stabilities of ES cells in the long term, resulting in karyotype abnormalities and impaired methylation of imprinted genes (Choi et al, 2017; Yagi et al, 2017) These changes eventually lead to reduced developmental potency of ES cells, which impairs their chimeric ability and germline competence. Another problem of current approaches to generate mouse models using gene-targeted ES cells is that it is time-consuming and labor-intensive: it usually takes 9 months to 1 year to obtain the mouse model by breeding. There is a high demand for developing a robust and fast approach to generate mouse models with sophisticated and precise genetic modifications
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