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Bovine inner cell mass (ICM) cells as donor nuclei in the production of nuclear transfer embryos
Theriogenology
39:242,
1993
BOVINE INNER CELL MASS (ICM) CELLS AS DONOR NUCLEI IN THE PRODUCTION OF NUCLEAR TRANSFER EMBRYOS C.L. Keefer, R. Koppang, A.M. Paprocki, P. Golueke, S. Stice, M.Maki-Laurila and L. Matthews ABS Specialty Genetics 6908 River Road DeForest, Wisconsin 53532 USA Embryos used in nuclear transfer procedures as the source for donor nuclei are commonly at the 32-64 cell stage. Navara & First (Biol. Reprod. 46(Sl): abst. 82, 1992) have shown that nuclear transfer of outside polarized cells of compact bovine morulae result in poor development. Their finding indicated that differentiation of the cells decreases the developmental potential of these embryonic cells. The objective of this study was to determine whether bovine inner cell mass (ICM) cells could be used as donor nuclei in the nuclear transfer procedure. Inner cell masses were isolated by immunosurgery from expanded day 7 to 9 blastocysts which had been produced in vitro. The isolated ICMs were placed into empty zonae pellucidae to ease handling during nuclear transfer. The manipulation medium used for enucleation and for ICM separation contained 7.5 ,ug cytochalasin B (CB) per ml. CB loosened ICM cellular connections and the edge of the zona pellucida was used to pinch off individual ICM cells. Each individual ICM cell was transferred into an enucleated oocyte. Oocytes were checked for enucleation with Hoechst dye to ensure that all DNA was removed, thus eliminating the possibility for parthenogenetic development. ICM celloocyte units were fused by a brief electrical pulse (110 V DC, 15 psec in a 500 pm chamber) and the resulting zygotes placed into CR1 bovine embryo culture medium supplemented with amino acids (Rosenkrans & First. Therio. 35: abst. 266, 1991) and fetal calf serum. The nuclear transfer embryos were scored for development to the morula and blastocyst stages on Day 7 (day of fusion = 0). A total of 808 cells were transferred. Fusion was not determined owing to the small size of the ICM cells. Fifty-two (6%) developed to morulae and blastocysts. Twenty ICM derived blastocysts were transferred into recipients. Four were identified by ultrasound as being pregnant on day 28-35. Two pregnancies at 100 days and one pregnancy at 190 days remain ongoing at time of abstract submission. This study demonstrates that inner cell mass cells from bovine expanded blastocysts are, at least, pluripotent, as ICM cells following nuclear transfer to enucleated oocytes can direct normal embryonic and fetal development. Birth and development of the calves will demonstrate totipotency. Totipotency of ICM cells would permit the use of older embryos in the cloning process and would be encouraging for the development of embryonic stem cell lines in the bovine.
242
Copyright Q 1993 Butter-worth-Heinemann
39:242,
1993
BOVINE INNER CELL MASS (ICM) CELLS AS DONOR NUCLEI IN THE PRODUCTION OF NUCLEAR TRANSFER EMBRYOS C.L. Keefer, R. Koppang, A.M. Paprocki, P. Golueke, S. Stice, M.Maki-Laurila and L. Matthews ABS Specialty Genetics 6908 River Road DeForest, Wisconsin 53532 USA Embryos used in nuclear transfer procedures as the source for donor nuclei are commonly at the 32-64 cell stage. Navara & First (Biol. Reprod. 46(Sl): abst. 82, 1992) have shown that nuclear transfer of outside polarized cells of compact bovine morulae result in poor development. Their finding indicated that differentiation of the cells decreases the developmental potential of these embryonic cells. The objective of this study was to determine whether bovine inner cell mass (ICM) cells could be used as donor nuclei in the nuclear transfer procedure. Inner cell masses were isolated by immunosurgery from expanded day 7 to 9 blastocysts which had been produced in vitro. The isolated ICMs were placed into empty zonae pellucidae to ease handling during nuclear transfer. The manipulation medium used for enucleation and for ICM separation contained 7.5 ,ug cytochalasin B (CB) per ml. CB loosened ICM cellular connections and the edge of the zona pellucida was used to pinch off individual ICM cells. Each individual ICM cell was transferred into an enucleated oocyte. Oocytes were checked for enucleation with Hoechst dye to ensure that all DNA was removed, thus eliminating the possibility for parthenogenetic development. ICM celloocyte units were fused by a brief electrical pulse (110 V DC, 15 psec in a 500 pm chamber) and the resulting zygotes placed into CR1 bovine embryo culture medium supplemented with amino acids (Rosenkrans & First. Therio. 35: abst. 266, 1991) and fetal calf serum. The nuclear transfer embryos were scored for development to the morula and blastocyst stages on Day 7 (day of fusion = 0). A total of 808 cells were transferred. Fusion was not determined owing to the small size of the ICM cells. Fifty-two (6%) developed to morulae and blastocysts. Twenty ICM derived blastocysts were transferred into recipients. Four were identified by ultrasound as being pregnant on day 28-35. Two pregnancies at 100 days and one pregnancy at 190 days remain ongoing at time of abstract submission. This study demonstrates that inner cell mass cells from bovine expanded blastocysts are, at least, pluripotent, as ICM cells following nuclear transfer to enucleated oocytes can direct normal embryonic and fetal development. Birth and development of the calves will demonstrate totipotency. Totipotency of ICM cells would permit the use of older embryos in the cloning process and would be encouraging for the development of embryonic stem cell lines in the bovine.
242
Copyright Q 1993 Butter-worth-Heinemann