Rapid generation of gene-targeted EPS-derived mouse models through tetraploid complementation

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.

High concentration of Cas12a effector tolerates more mismatches on ssDNA

“…Over the past few years, the Clustered regularly interspaced short palindromic repeats/CRISPR‐associated (CRISPR/Cas) system has shown excellent performance in DNA editing 5‐8 and regulation, 9 and RNA editing 10 . This system can also be potentially used for nucleic‐acid detection 11‐14 .…”

Derivation of human extended pluripotent stem cells in feeder-free condition

“…In this study, we started with optimizing the transition and maintenance condition of human EPSC in feeder-free, and then characterized the molecular and biological properties of the converted EPSCs in detail. Our data demonstrated the human ffEPSCs was recently shown to be able to greatly advance the efficiency in generating genetargeting mouse models [28] , indicating the significance and broad application. Second, OCT4 and NANOG are functionally expressed in both naive and primed pluripotent ESCs and iPSCs, but not or lower expressed in totipotent-like cells including either early human embryos before later morula stage [16] or 2-cell-like cells existing in mouse ESC cultures [6] .…”

Cell Signaling Pathway Reporters in Adult Hematopoietic Stem Cells

“…CRISPR-Cas9 transgenesis is thought to make a breakthrough in rapid and effective generation of transgenic models, not to mention it would settle the dilemma of randomly inserted DNA binding sites. Recently, an efficient method to generate gene-targeted mouse models was proposed through tetraploid complementation in EPS cells in just 2 months’ time [ 68 ].…”