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With the aid of a dissecting microscope, the extraembryonic tissue was manually removed from embryos in phosphate buffered saline (PBS). To dissociate the germlayers, embryos were subsequently placed in 500 uL of 1% trypsin in Hanks solution. After 30 minutes of incubation on ice, the digestion was stopped with the addition of 1 mL of 20% fetal bovine serum (FBS) in Hanks solution plus a small amount of DNase I powder. Tungsten needles or fine forceps were used to tease away the whole endoderm from other two germ layers, and then was collected in tubes containing Trizol.