Construction of aggregation chimaeras from 8-cell mouse embryos stored for several years in liquid nitrogen

Eight-cell embryos of 7 different inbred strains frozen and stored for 2 to 4 years in liquid nitrogen were used as the source of embryos and individual 1/8-blastomeres for construction of different types of aggregation chimaeras. Overall survival of stored embryos, evaluated by in vitro culture, ranged from 63% in A/By to 85% in C57BL/10By embryos. Chimaeric embryos prepared by aggregation of frozen/thawed zona-free 8-cell embryos continued in normal development through expanding blastocysts (77% to 93%) to in vitro implantations. Construction of directed chimaeras by total rebuilding of 8-cell embryos previously stored in liquid nitrogen was successful and resulted in normal development. Of the aggregates made by regular mixing of 1/8-blastomeres obtained from BALB/c and C57BL/6J embryos thawed from liquid nitrogen, 90% reached the blastocyst stage and 77% implanted after 48 h and 96 h of in vitro culture, respectively. Addition of 1/8 blastomeres from frozen/thawed BALB/c embryos to four 1/8-like blastomeres from nuclear transplantation experiments documented, as all of the 30 constructed aggregates formed expanding blastocysts within 48 h of in vitro culture, the possibility of using embryos from embryo banks for the rescue of products of genetic manipulations at the embryo level.