An alternative set of mouse models, beyond the naturally occurring lupus strains discussed above, consists of knockout or transgenic genetic modifications that profoundly affect a single susceptibility gene.
The hESC medium consisted of KnockOut DMEM (KO DMEM) supplemented with 20% KnockOut serum replacement (KOSR), 1 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 1% non-essential amino acids (100x stock) and 0.1 mM β-mercaptoethanol.
The medium used for derivation and culture of the first four established hESC lines consisted of Knockout Dulbecco's modified Eagle's medium (GibcoBRL, Life Technologies), supplemented with 2 nmol/l L-glutamine, 20% FCS (R&D, Sweden), 0.1 mmol/l b-mercaptoethanol (Gibco), 1% non-essential amino acids (Gibco), and recombinant human LIF, 1 ml/ml (Chemicon, UK).
For cardiomyocyte differentiation, the hEBs were plated in gelatin-coated cell culture dishes in differentiation medium consisting of Knockout DMEM (Invitrogen), 20% FBS (Invitrogen), 1% non-essential amino acids, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% penicillin/streptomycin for 3 weeks.
The resulting clumps were added to the non-tissue culture treated plastic petri dish and cultured in suspension for the indicated periods in hEB medium consisting of Knockout DMEM (Invitrogen), 20% FBS (Invitrogen), 1% non-essential amino acids, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% penicillin/streptomycin (Fig. 1Ai).
hES medium consisted of knockout Dulbecco's modified Eagle's medium (Invitrogen GIBCO), 10% KO-Serum Replacement (Invitrogen GIBCO), 0.5% albumin (cat. no.
Clones of iPSC were cultured on irradiated MEF (5 × 10 cells/cm) in ESC culture medium that consisted of Knockout™ DMEM supplemented with 15 % Knockout™ serum replacement, 0.5%% penicillin/streptomycin, 0.1 mmol/L non-essential amino acids, 0.2 mmol/L l-glutamine, 0.1 mmol/L mercaptoethanol (Invitrogen, CA, USA), and 10 U ml−1 leukemia inhibitory factor (LIF; Millipore, CA, USA).
Approximately 24 hours after second viral infection, cells were switched to ESC media consisting of knockout DMEM supplemented with 20% knockout serum replacement, 1 mM L-glutamine, 1% non-essential amino acid, 0.1 mM ß-mercaptoethanol and 10 ng/ml human basic fibroblast growth factor (bFGF; Invitrogen).
The neuronal and glial differentiation medium consists of the Knockout medium (Invitrogen) supplemented with 15% Knockout Serum SR, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol (all from Invitrogen) and depleted of LIF.
The top two teams from each group are then drawn into the knockout stage, which consists of two-legged knockout ties.
