Mice cloned from white adipose tissue-derived cells

Isolation of different cells from adipose tissue was performed according to the previous reports (Rodeheffer et al., 2008; Sugii et al., 2010). Briefly, the inguinal fat pads of adult male B6D2F1 (8-12 weeks of age) mice were harvested, washed several times with phosphate-buffered saline (PBS) and excised into small pieces. The tissues were digested with 0.1% type I collagenase (Sigma) for 50 min at 37°C shaking, followed by adding equal volume of Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) with 10% fetal bovine serum (FBS; Gibico) to neutralize enzyme activity. The cell suspensions were centrifuged at 400 g for 8 min and the supernatant was removed. The pelleted cells, so-called SVF cells, were suspended with PBS containing 2% FBS and were incubated with antibodies. Antibodies used in this study were purchased from eBioscience unless otherwise stated, including CD45-APC-Cy7, Terr119-FITC, CD31-biotin (BD Biosciences), PE-Texas Red (BD Biosciences), CD140a-PE, CD140b-PE, CD105-PE, CD13-FITC, Sca-1-PE and CD34-APC. Antibody incubations were performed on ice for 20 min. Samples were sorted on a BD FACSAria II cell sorter and analyzed on a BD Calibur flow cytometer, each equipped with BD FACSDiva Software. The cells were separated on the basis of the cell-surface markers indicated. Sorted Lin cells and CD45 + cells were suspended in HEPES-buffered CZB (HCZB; Sigma) medium with 2% polyvinylpyrrolidone (PVP; Sigma) for nuclear transfer (NT).