Saluki Skin Cells for Nuclear Transfer

To obtain cells for nuclear transfer; a skin biopsy was obtained from a living Saluki. These clones would later be used as a syngeneic control for UDC SCNT. After mechanical and enzymatic dissociation of the tissue (Hossein, 2009 #2), cells were allowed to attach to a culture dish (Product No. 353002, Falcon^®, Corning) in Dulbecco modified Eagle medium (Cat. #11995065, DMEM high glucose pyruvate, GibcoTM) with 10% fetal bovine serum (Cat. #16000044, FBS, GibcoTM) at 37 °C in an atmosphere of 5% CO2 and air. Media was changed every 48 hrs until the cellular monolayer reached approximately 80% confluence. UDCs used in the cloning experiments were obtained, as described above (UDCs), from two cloned saluki pups of the same donor origin. Both explants and UDCs were cultured until they approached 90% confluence, trypsinized and reconstituted at concentrations of approximately 1 x 10⁶ cells per mL, then cryopreserved in cryovials containing DMEM + 20% FBS +10% DMSO. Prior to cryopreservation cells were taken in the same manner to be used as donor cells for SCNT. After cell populations reached near confluence, cells were used for SCNT (Nuclear Transfer).