Cloned Mice: MATERIALS AND METHODS(2)

METHODS(2)Immunocytochemistry

Oocytes were fixed and immunostained for microtubules and microfilaments using a modification of the method described by Messinger and Albertini. Briefly, oocytes were fixed and extracted for 30 min at 37°C in a microtubule-stabilizing buffer (0.1 M Pipes [pH 6.9], 5 mM MgCl26H2O, and 2.5 mM EGTA) containing 2% formaldehyde, 0.5% Triton X-100, 50% deuterium oxide, and 1 mM of dithiothreitol. Oocytes were then washed three times in a blocking solution of PBS containing 10% normal goat serum (NGS), 0.1% Triton X-100, and 0.02% sodium azide before being stored at 4°C until processing for immunocytochemis-try. Oocytes were incubated with fluorescein isothiocyanate-conjugated anti-a-tubulin antibody (final dilution, 1:500; Sigma) and rhodamine phal-loidin (1:4000; Molecular Probes, Eugene, OR) in a blocking solution of PBS containing 5% NGS in the dark at 37°C for 1 h. After three washes in the 10% NGS blocking solution, oocytes were mounted in Vectashield (Vector Laboratories Ltd., Peterborough, U.K.) containing 5 |xg/ml of 4′,6-diamidino-2-phenylindole and then assessed. Labeled oocytes were viewed using a Zeiss Axiovert S 100 photomicroscope (Carl Zeiss, Welwyn Garden City, U.K.) equipped with fluorescein (Zeiss 487709), Texas Red (Zeiss 487714), and Hoechst 33342 (Zeiss 487702) selective filter sets and a 50-W mercury arc lamp. Images were acquired using Kinetic Imaging System (Imaging Associates Ltd., Thame, U.K.) whitening gel

ES Cell Culture

The hypoxanthine phosphoribosyltransferase-deficient ES cell line HM-1, derived from the inbred mouse strain 129/Ola, was kindly supplied by Dr. Ed Gallagher (Roslin Institute, Scotland, U.K.) at passage 19. Previously, the capacity of this cell line to yield chimeric animals and germ-line transmission was confirmed by its injection into mouse blastocysts. HM-1 ES cells were cultured in Glasgow modified Eagle medium supplemented with 15% fetal calf serum (Globepharm, Guildford, Surrey, U.K.), 1000 U/ml of leukemia inhibitory factor, 1% L-glutamine, 1% sodium pyruvate, 1% modified Eagle medium nonessential amino acids, and 0.22% р-mercaptoethanol. Cells were serum deprived for 18-20 h by reducing the concentration of serum to 5% before their use in nuclear transfer experiments. Within 1 h of being required for nuclear transfer, ES cells were lifted using trypsin-EGTA (TEG) medium consisting of 0.25% trypsin (Invitrogen Life Technologies Ltd., Paisley, U.K.), 1.1 mM EGTA, 0.01% polyvinyl alcohol (PVA; molecular weight, 30 000-70 000), 108 mM NaCl, 0.67 mM Na2HPO4-2H2O, 1.6 mM KH2PO4, 4.5 mM KCl, 5 mM D-glucose, and 22.3 mM Tris, at pH 7.6. With the exception of EGTA and PVA, all salts in TEG medium were BDH Analar grade (BDH Laboratory Supplies, Poole, U.K.).

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