Preparation of Animals with a High Degree of Chimerism by One-Step Coculture of Embryonic Stem Cells and Preimplantation Embryos

摘要

A single-step coculture procedure has been developed that can generate chimeric mice with high efficiency and reproducibili-ty. The procedure involves culture of embryonic stem (ES) cells with 8- to 16-cell embryos in microwells to provide conditionsfor effective cell-cell-embryo contact A suspension of ES cells is layered over the microwells, followed by transfer of an embryo without zona pellucida into each microwell Following overnight culture, the blastocysts are transferred into pseudopregnantrecipients. The method has several advantages due to its simplicity and reproducibility: (i) Over 90% of ES-cell contribution in new-bar ns can be obtained frequently and most of the chimeras display germ-line transmission (ii) The procedure does not require specialized skills or expensive instruments, (iii) All the steps of embryo manipulation can be completed in a relatively short period of time; therefore, a large number of embryos can be manipulated simultaneously, The method was tested with three independent ES cell lines and two different strains of mice with similar results. The technique may be an alternative to microinjection of DNA into zygotes to prepare transgenic lines of small and large mammalian species.