Cell culture and DmES cells in tissue culture
C3H/10T1/2 (10T1/2) cells were grown in Deulbecco's modified eagle medium containing 10% fetal bovine serum (FBS), 50 μg/ml of penicillin and streptomycin at 37°C and 5% CO2. Mouse embryonic stem (mES) cells were grown in gelatinized flasks in Glasgow minimum essential medium (GMEM) containing 20% FBS (qualified for ES cell culture), 50 ug/ml of penicillin and streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 0.1 mM 2-mercaptoethanol and 1000 U/ml leukaemia inhibitory factor. These mES cells are feeder free. mES cells were differentiated into neural cells in differentiation medium (GMEM containing 20% FBS, 50 μg/ml of penicillin and streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, and 5 μM retinoic acid). mES cells usually differentiated into neural cells, neurons and glial cells, after 3 days in the differentiation medium. These differentiated mES cells were maintained in the differentiation medium until they were harvested for nuclear injection.
Oocyte preparation and injection and cell permeabilization
Oocytes of X. laevis were prepared and treated as described before . For these experiments we have aimed to inject 200 nuclei per oocyte. We find that 80% injected nuclei are usually deposited in the GV.
Gene expression analysis on transplanted somatic cell nuclei by RT-PCR
RNA from four injected oocytes was isolated using an RNA isolation kit (Qiagen, CA, USA). cDNA was generated using Superscript III reverse transcriptase (Invitrogen, CA, USA). We used gene specific primers for synthesizing first strand DNA. We added 10 mM dithiothreitol, 1 mM dNTPs, 0.2 mg/ml bovine serum albumin and 40 units RNase inhibitors and incubated them at 42°C for 1 h. A part of the sample was diluted (1:10, vol/vol) by DNase/RNase free water and used immediately for polymerase chain reaction (PCR).
PCR was performed for 35 cycles under conditions of 95°C (30 s), 58°C (30 s) and 72°C (90 s) following a preheating step at 95°C for 15 min to amplify embryo-expressed genes but was performed for 25 cycles to analyse the GAPDH gene. Hot Start Taq polymerase was used in the presence of 25 mM MgSO4, 10 mM dNTPs and 10 nM gene specific primers in the buffer supplied by a company. Q-solution was added to the samples to amplify Oct4 or Sox2. PCR products were separated by electrophoresis on a 1.5% agarose gel. The primer sequences for this study are
Sox2 forward: GGAGTGGAAACTTTTGTCCGAGAC, Sox2 reverse: TGGAGTGGGAGGAAGAGGTAACC, Oct4 forward: GTGAGCCGTCTTTCCACCAG, Oct4 reverse: TTCTCCAACTTCACGGCATT, Sall4 forward: GGGGCTAAAATTTCCCAACT, Sall4 reverse: CTCCTCCCAGTTGATGTGCT, β-globin forward: GCTGGTTGTCTACCCTTGGA, β-globin reverse: ATCCACATGCAGCTTGTCAC, GAPDH forward: TCAACGACCCCTTCATTGAC, GAPDH reverse: ATGCAGGGATGATGTTCTGG.
Immunofluorescent staining of injected nuclei
Injected oocytes were collected at 0, 12, 24, 48 or 72 h after nuclear transfer. The injected oocytes at zero hour were dissected within 1 min after the injection. Approximately 50 injected oocytes were harvested at each time point. Germinal vesicles from the injected oocytes were separated in isolation buffer (20 mM Tris/HCl pH7.5, 0.5 mM MgSO4, 140 mM KCl). The isolated GVs which contained injected somatic cell nuclei were transferred to 1 ml of 4% paraformaldehyde solution and fixed for 10 min. After the paraformaldehyde solution was removed, they were permeabilized in 1 ml of permeabilization buffer (0.5% Triton X-100 solution in phosphate buffered saline (PBS) for 5 min and washed three times with 1 ml of Washing/Blocking buffer (5% FBS, 0.2% Tween-20 in PBS). They were incubated in the same solution for at least 1 h for blocking. The injected somatic cell nuclei in GVs were resuspended in approximately 50 μl of washing/blocking buffer and stained by primary antibodies against the desired antigens (typically 1/100 or 1/200 dilution, depending on antibodies) over night. After staining, they were washed three times with washing/blocking buffer to remove excess primary antibodies and resuspended in 50 μl of washing/blocking buffer. They were then stained by secondary antibodies which were conjugated with alexa fluor (Molecular Probes, Oregon, USA; 1/200 dilution). They were washed three times with washing/blocking buffer and counterstained with DAPI (Invitrogen). Antibodies were provided by AB and TK, or purchased from Abcam.
Western blotting of histones in nuclei from differentiated cells injected into germinal vesicles of X. oocytes or in nuclei treated with high speed X. oocyte extracts
Injected oocytes were collected and dissected in isolation buffer described above. The isolated GVs with injected cell nuclei were transferred to a tube with 1 ml of non-denaturing washing buffer (150 mM Tris-HCl ph7.5, 50 mM NaCl, 150 mM KCl). The GVs were vigorously vortexed for about 5 s in order to destroy GV membranes so that transplanted 10T 1/2 nuclei could be separated from the GVs. The nuclei were pelleted by a 16,000 g centrifugation for 3 min at room temperature. The nuclear pellets were washed twice with the non-denaturing washing buffer to remove any contaminants from GVs. The nuclear pellets recovered from GVs were lysed in high salt radioimmunopreciptation buffer (25 mM Tris-HCl pH7.6, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 2.5 M NaCl) with a protease inhibitor cocktail (Roche). The pellets were vigorously vortexed in the buffer at 4°C for 10 min and membranes were separated by a 13.2 krpm centrifugation at 4°C for 30 min.
Approximately 20,000 10T 1/2 cell nuclei were incubated in 20 μl of high speed oocyte extracts or in buffer for 0, 1 or 2 h at 18°C (the concentration of 10T1/2 cell nuclei in extracts was 1,000/μl). The nuclei were spun down with a 10 k rpm centrifugation at room temperature after the desired incubation. The nuclear pellets were washed with non-denaturing buffer three times with a 16,000 g centrifugation after each washing. These lysates were used for Western blotting.
ChIP analysis on cell nuclei recovered from X. laevis germinal vesicles or injected oocytes
Approximately 200 10T1/2 cell nuclei or DmES cell nuclei were injected into X. laevis oocyte GVs and were collected at the desired times such as 0, 24, 48 or 72 h after nuclear transfer. Whole injected oocytes were lysed in a MNase digestion buffer (0.32 M Sucrose, 50 mM Tris-HCl pH 9.5, 4 mM MgCl2, 1 mM CaCl2, 0.1 mM phenylmethanesulphonylfuoride). The pH of Tris-HCl in this modified MNase digestion buffer is high because oocyte lysates are acidic and we used a limited quantity of the buffer so as not to dilute histones in samples (50 whole injected oocytes in 100 μl buffer). Therefore, Tris-HCl pH 9.5 was required to maintain oocyte lysates containing injected nuclei at around pH 7.5. The chromosomes in nuclear pellets were digested by micrococcal nuclease (MNase, 0.1 units/sample) at 18°C for 10 min in order to obtain mononucleosomes. The cell permeabilization step was omitted because these cells were already permeabilized before injection. 1 μl of 0.5 M EDTA and 1 μl of NP-40 was added to a 100 μl sample solution in order to stop micrococcal nuclease activities. The pellets were shaken gently for 30 min at 4°C to lyse nuclei completely. Samples were spun down and only supernatants were collected as a soluble chromatin fraction. Primary antibodies were added to the chromatin solution and incubated at 4°C over night. Non-immune rabbit IgG was used in the preliminary experiments. ChIP by histone H3 antibodies was used as a histone precipitation control instead of conducting sequential ChIPs. Protein A conjugated magnetic beads were added to the solution in order to precipitate the histone-antibody complexes. The histone-antibody complexes were washed extensively nine times with washing buffers containing a different concentration of salt (first washing buffer, three times with 75 mM NaCl; second washing buffer, three times with 125 mM NaCl; third washing buffer, three times with 175 mM NaCl in Tris-HCl pH 7.5 and 10 mM EDTA). The histone-antibody complexes were then digested by proteinase K overnight in elution buffer (50 mM Tis-HCl pH 7.5, 50 mM NaCl, 0.1 mM PMSF, 5 mM EDTA, 1% SDS). The free DNA was eluted in elution buffer. The eluted DNA (ChIP DNA) was purified by phenol chloroform extraction and ethanol precipitation method.
PCR was performed by real-time PCR (Applied Biosystems, CA, USA) for 40 cycles under conditions of 95°C (15 s), 58°C (60 s), and 60°C (60 s) followed by steps at 95°C for 15 s, 60°C for 60 s, 95°C for 15 s and 60°C for 15 s. The primer sequences for this study are from references  and .
Sox2 promoter region forward: CCATCCACCCTTATGTATCCAAG, Sox2 promoter region reverse: CGAAGGAAGTGGGTAAACAGCAC, Sox2 downstream regulatory region forward: CAGGTTCCCCTCTAATTAATGC, Sox2 downstream regulatory region reverse: CTGTGCTCATTACCACGTGAA, Sox2 gene coding region forward: GGAGCAACGGCAGCTA, Sox2 gene coding region reverse: GTAGCGGTGCATCGGT, Oct4 promoter region1 forward: GGCTCTCCAGAGGATGGCTGAG, Oct4 promoter region1 reverse: TCGGATGCCCCATCGCA, Oct4 gene coding region forward: CCTGCAGAAGGAGCTAGAACA, Oct4 gene coding region reverse: TGTGGAGAAGCAGCTCCTAAG, Sall4 promoter region forward: ATGCTGGGCCTTGTAGTCC, Sall4 promoter region reverse: ATCTGAGCCCGGATGCTAAT, β-globin promoter region forward: CTGCTCACACAGGATAGAGAGGG, β-globin promoter region reverse: GCAAATGTGAGGAGCAACTGTC, β-globin gene coding region forward: TCTACAGTTATGTTGATGGTTCTTCCA, β-globin gene coding region reverse: CAGGACAATCACGATCATATTGC.
The analysis of ChIP results was made as follows. The immediate results of real-time PCR were corrected by subtraction of the IgG background. These values were then adjusted so that the number 1.0 was allocated in each case to the T0 point, other values being changed in proportion. Finally the results were corrected by dividing each value by that obtained for the H3 histone control.
Immunodepletion of Aurora B from oocyte extracts
Fifty microlitres of antibodies against Aurora B (Santa Cruz, CA, USA) or non-immune mouse IgG were added to 100 μl of oocyte extracts for 1 h at 4°C and protein A beads (Invitrogen) were added to precipitate antibodies for 30 min.