33development of Bovine Embryos Cloned With Fetal Fibroblasts Arrested At G0/G1 Phase by Different Treatments

Abstract

The ability to produce cloned livestock using postmortem tissue could incorporate an additional application into the field of nuclear transfer. This study examined the feasibility of producing cloned cattle using a primary cell line established from a postmortem beef carcass. A market beef heifer processed at a USDA-certified slaughterhouse was used to develop a primary somatic cell line. Tissue samples were taken from the kidney and forelimb regions either 1) immediately following slaughter (fresh) or 2) 48 h postslaughter (cooled) where the carcass was housed at 2 to 4 • C. Tissue was removed and placed on ice in PBS + 5.0% (v:v) penicillin/streptomycin. A primary culture was established using standard techniques and cultured in supplemented DMEM F-12 medium. Once established, cells were trypsinized and either frozen or continually passaged. Cells used for nuclear transfer (NT) were passaged (48 h before use) and cultured with 15 µM roscovitine roughly 24 h prior to nuclear transfer. Cells were approximately 80% confluent and between passage numbers 1 and 11 at the time of NT. Selected slaughterhouse-derived oocytes were matured in supplemented TCM 199 medium for 18-20 h at 39 • C in 5.0% CO 2 and air. Mature Metaphase II oocytes were vortexed and stained with Hoechst 33342 to help with chromatin removal. Following enucleation, roscovitine-treated carcass cells were placed in the perivitelline space of the oocyte. Reconstructed NT embryos were fused in Zimmermann's medium and pulsed using needle-like electrodes. This was followed by activation using a combination of calcium ionophore (5 µM), cytochalasin D (5 µg mL −1 ), and cycloheximide (10 µg mL −1 ) in TCM + 10% FBS. Fused NT embryos were cultured in 50-µL drops of BARC medium (USDA, Beltsville, MD) for 7 days at 39 • C in a 5% CO 2 , 5% O 2 and 90% N 2 environment. Embryo development for all four groups (Table 1) was assessed with blastocysts (grade 1 or 2) being transferred into recipient cows 7 days post-estrus. Cleavage rates were not significantly different between groups, and the use of either fresh or cooled cells did not impact blastocyst formation. However, there was a significant difference (P = 0.05) in % blastocyst based on the source of the donor cell. Overall, one live calf resulted from 34 transferred NTs produced using kidney cells taken from a 48 h cooled carcass. These results display the feasibility of producing cloned calves from cells collected post mortem, which ultimately could be used as a tool to select breeding bulls based on their own steer carcass characteristics.