Nuclear Transplantation in the Bovine Embryo: Assessment of Donor Nuclei and Recipient Oocyt14

Abstract

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammalian nuclear transfer

“…All clones were derived by nuclear transplantation using nuclei from blastomeres as donors (Willadsen, 1986;Prather et al, 1987Prather et al, , 1989. Importantly, these researchers used oocytes rather than zygotes as recipients.…”

“…For normal development to proceed, it is crucial that, in a Briggs et al, 1952Briggs et al, 1962 Nuclear transfer-derived frogs Gurdon, 1962Gurdon, 1975 Rabbit nuclear transfer Bromhall, 1975Bromhall, 1979 Nuclear transplantation in fish Gasaryan et al, 1979Gasaryan et al, 1981 Derivation of mouse ES cells Evans and Kaufman, 1981;Martin, 1981Martin, 1983 Nuclear transplantation in the mouse Solter, 1983 1986 Lambs cloned from blastomeres Willadsen, 1986Willadsen, 1987 Cattle cloned from blastomeres Prather et al, 1987Prather et al, 1989 Pigs cloned from blastomeres Prather et al, 1989Prather et al, 1994 Calves cloned from ICM cells First, 1994 1996 Sheep cloned from embryonic cell line Campbell et al, 1996 First mammal cloned from adult cell (sheep "Dolly") Wilmut et al, 1997Wilmut et al, 1998 Derivation of human ES cells Thomson et al, 1998Thomson et al, 2000 Mouse nuclear transfer-derived ES cells Munsie et al, 2000Munsie et al, 2002 Terminally differentiated cells cloned Hochedlinger et al, 2002aHochedlinger et al, 2002 First therapeutic application in mice Rideout et al, 2002 a ES, embryonic stem; ICM, inner cell mass. This table summarizes a number of selected highlights starting from the first demonstration of the NT technique by Briggs and King to the first "proof of principle" study in mice showing the therapeutic potential of the technique.…”

Correlation of Developmental Differences of Nuclear Transfer Embryos Cells to the Methylation Profiles of Nuclear Transfer Donor Cells in Swine

“…Many cells types have been used as donor cells to produce live, fertile offspring. The donor cells could be classified as either: (1) blastomeres from the early embryo, (2) embryonic stem cells (ES), (3) somatic stem cells or (4) fully differentiated somatic cells. The use of blastomeres as donor cells were successfully used to generate the first mammalian clones, [1][2][3] Embryonic stem cells have been successfully used as donor cells in the mouse.…”

“…The donor cells could be classified as either: (1) blastomeres from the early embryo, (2) embryonic stem cells (ES), (3) somatic stem cells or (4) fully differentiated somatic cells. The use of blastomeres as donor cells were successfully used to generate the first mammalian clones, [1][2][3] Embryonic stem cells have been successfully used as donor cells in the mouse. 4,5 Higher rates of development have been reported when using ES cells as the donor cells compared to somatic donor cells.…”

Transgenic technology in farm animals - progress and perspectives

“…Initial nuclear transfer experiments in livestock used blastomeres from pre-implantation embryos as nuclear donors (Willadsen, 1986;Prather et al 1987Prather et al , 1989, limiting practical applications in biotechnology. However, pioneering experiments carried out at the Roslin Institute, Edinburgh, demonstrated first that ovine embryonic cells can be expanded in culture before using them for nuclear transfer (Campbell et al 1996), and later that not only cells of embryonic origin, but also cells derived from fetal or adult tissues can be Gene transfer in farm animals Exp.…”