For production of recombinant proteins, cDNAs encoding the catalytic domains of mouse Tet1 (aa1367–2039), Tet2 (aa916–1921), Tet3 (aa697–1668), and their corresponding catalytic mutants (Ito et al., 2010
) are cloned into a modified pFastBac-HTb (Invitrogen) insect cell expression vector inframe with a FLAG tag at the N-terminus. Constructs are transformed into DH10Bac Escherichia coli
(Invitrogen) following manufacturer’s instructions to generate bacmid DNA. Since recombinant bacmid DNA is ≥ 135 kbp, most silica-gel-membrane-based plasmid miniprep kits are not suitable for bacmid DNA isolation, anion-exchange-resin-based kits should be used instead (e.g., PureLink HiPure Plasmid Miniprep Kit (Invitrogen)). Alternatively, we found that crude purification of bacmid DNA using conventional alkaline lysis followed by ethanol precipitation could also give satisfying results in the subsequent Sf9 cell transfections. To verify the successful transposition of Tet cDNAs to bacmid DNA, polymerase chain reaction (PCR) analysis is performed with pUC/M13 forward (5′-CCCAGTCACGACGTTGTAAAACG-3′) and reverse (5′-AGCGGATAACAATTTCACACAGG-3′) primers. A PCR band of ~2400 bp+ the size of the insert indicates successful transposition, otherwise, an ~350 bp band will show up. Once correct bacmid DNA is generated, baculovirus can be produced in Sf9 cells.
Sf9 cells are maintained at a density of 0.5–6×106 cells/ml in Sf-900 II SFM medium (Invitrogen) containing 10% fetal bovine serum in a spinner flask at 27 °C. Before transfection, cells are seeded in a six-well plate with 9×105 cells/well and allowed to attach for 1 h at 27 °C. Dilute 1 μg of bacmid DNA (or the amount from 100 μl of bacteria if crude bacmid DNA is used) and 8 μl of Cellfectin II (Invitrogen) separately with 100 μl of Grace’s Insect Medium (unsupplemented). Combine the diluted bacmid with diluted Cellfectin II reagent and incubate at room temperature for 30 min. While the transfection complexes are forming, remove the medium from the plate and wash cells once with 2 ml of Grace’s Insect Medium (unsupplemented). Remove the wash medium, add 800 μl of Grace’s Insect Medium (unsupplemented) to the transfection mixture and gently add the mixture onto the cells immediately. Incubate cells for 5 h at 27 °C before replacing the transfection mixture with 2 ml of complete growth medium (Sf-900 II SFM medium containing 10% fetal bovine serum). After 96 h, collect the medium and centrifuge at 500×g for 5 min to remove floating cells. Transfer the supernatant containing baculovirus to a new tube and store the P1 viral stock at 4 °C protected from light. To verify the successful production of virus, expression of recombinant Tet proteins in the transfected cells can be examined by Western blot, although the expression may be weak at this stage. To amplify the P1 viral stock, seed 2×106 cells/well in a six-well plate, add 50 μl of P1 viral stock, and incubate for 72 h. Collect the P2 viral stock as per the P1 stock. We usually further amplify the P2 viral stock to generate P3 viral stock of higher titer. Seed 9×106 cells/dish in a 10 cm dish, add 250 μl of P2 viral stock, and incubate for 72 h. The titer of the P3 viral stock is usually ~2×108 pfu/ml.
To express the Tet proteins, prepare 1 l of Sf9 cells in mid log growth phase (1.5–2×106 cells/ml) in a spinner flask, infect the cells with 20 ml P3 viral stock (the multiplicity of infection (MOI) is ~2) for 72 h. Cells are collected and washed once with ice-cold phosphate buffered saline. Cell pellet is then resuspended in 40 ml of LysisBuffer F (50 mM HEPES, 500 mM NaCl,2 mM MgCl2, 2 mM DTT, 0.2% NP-40, 20% glycerol, 1× protease inhibitors without EDTA (Roche), pH 8.0) and transferred to a Dounce homogenizer with a type A pestle. Homogenize slowly for 30 times over a 30-min period on ice and centrifuge the cell lysate at 14,000 ×g for 20 min at 4 °C. While centrifuging the cell lysate, wash 1 ml of 50% slurry of FLAG M2 beads (Sigma) with 20 ml Lysis Buffer F in a 50 ml conical tube, spin at 500×g for 5 min, remove the supernatant. Transfer the supernatant of the cell lysate to the tube and rotate at 4 °C for 3 h. After the incubation, collect beads by spinning at 500×g for 5 min at 4 °C, wash beads with 30 ml Wash Buffer (50 mM HEPES, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 20% glycerol) for three times, and elute the protein with 500 μl Wash Buffer containing 200 μg/ml 3×FLAG peptide (Sigma) by rotating at 4 °C for 30 min. Aliquot the eluted protein and store at −80 °C. The purity of the eluted protein can be determined by Coomassie staining of an SDS-PAGE.