PREPARATION OF ANIMALS WITH A HIGH-DEGREE OF CHIMERISM BY ONE-STEP COCULTURE OF EMBRYONIC STEM-CELLS AND PREIMPLANTATION EMBRYOS

A single-step coculture procedure has been developed that can generate chimeric mice with high efficiency and reproducibility. The procedure involves culture embryonic stem (ES) cells with 8- to 16-cell embryos in microwells to provide conditions for effective cell-cell-embryo co ntact. A suspension of ES cells is layered over the microwells, follow ed by transfer of an embryo without zona pellucida into each microwell . Following overnight culture, the blastocysts are transferred into ps eudopregnant recipients. The method has several advantages due to ifs simplicity and reproducibility: (i) Over 90% of ES-cell contribution i n newborns can be obtained frequently and most of the chimeras display germ-line transmission. (ii) The procedure does not require specializ ed skills or expensive instruments. (iii) All the steps of embryo mani pulation can be completed in a relatively short peri od of time; there fore, a large number of embryos can be manipulated simultaneously The Method was tested with three independent ES cell lines and two differe nt strains of mice with similar results. The technique may be an alter native to microinjection of DNA into zygotes to prepare transgenic lin es of small and large mammalian species.