pMT-hFIX/M

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	All constructs along with pCoHygro plasmid were transfected into Drosophila S2 cells (Invitrogen, Lot no; 769,915) employing the calcium phosphate co-precipitation method with minor modifications [19]. After 48 h, the cells were cultured in 300 μg of hygromycin B/ml. To select the clones stably expressing FIX, the single cell strategy was employed [20].	To assess the functional rFIX expression, stable S2 cells were induced with 0.5 mM CuSO4 in the presence of 6 μg/ml of vitamin K1. Total FIX expression was quantified in conditioned media employing a FIX-specific antigen assay (ELISA) following the manufacturer’s instructions (Asserachrom, hFIX: Ag). The FIX clotting activity was determined employing a modified one-stage aPTT assay specific for FIX as described previously [18].

Protocol tips
All constructs along with pCoHygro plasmid were transfected into Drosophila S2 cells (Invitrogen, Lot no; 769,915) employing the calcium phosphate co-precipitation method with minor modifications [19]. After 48 h, the cells were cultured in 300 μg of hygromycin B/ml. To select the clones stably expressing FIX, the single cell strategy was employed [20].
Downstream tips
To assess the functional rFIX expression, stable S2 cells were induced with 0.5 mM CuSO4 in the presence of 6 μg/ml of vitamin K1. Total FIX expression was quantified in conditioned media employing a FIX-specific antigen assay (ELISA) following the manufacturer’s instructions (Asserachrom, hFIX: Ag). The FIX clotting activity was determined employing a modified one-stage aPTT assay specific for FIX as described previously [18].

Publication protocol

All constructs along with pCoHygro plasmid were transfected into Drosophila S2 cells (Invitrogen, Lot no; 769,915) employing the calcium phosphate co-precipitation method with minor modifications [19]. After 48 h, the cells were cultured in 300 μg of hygromycin B/ml. To select the clones stably expressing FIX, the single cell strategy was employed [20]. In brief, approximately 150 × 106 parental S2 cells were treated for 4 h with mitomycin C to induce cell cycle arrest, upon which these feeder cells were plated in 24-well plates at 3 × 106 cells/ml [21]. Subsequently, the FIX-transfected cells were added at 1 cell/well. After approximately two weeks, the single cell-derived clones were screened for FIX expression.
To assess the functional rFIX expression, stable S2 cells were induced with 0.5 mM CuSO4 in the presence of 6 μg/ml of vitamin K1. Total FIX expression was quantified in conditioned media employing a FIX-specific antigen assay (ELISA) following the manufacturer’s instructions (Asserachrom, hFIX: Ag). The FIX clotting activity was determined employing a modified one-stage aPTT assay specific for FIX as described previously [18]. For both analyses, FIX levels were corrected for measurements obtained using conditioned media from non-transfected cells. Reference curves of normal pooled plasma (provided by Hashemi Nezhad Hospital, Iran) were used to calculate the equivalent FIX concentration and clotting activity, respectively. By definition, one unit of FIX activity corresponds to the amount of FIX in 1 ml of normal plasma (~ 5 μg/ml).

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