mRNA steady-state levels, translation, and secretion of normal and mutated fVIII by transiently transfected CHO cells
| fVIII Gene . | mRNA3-150 . | Translation3-151 . | Translation/mRNA . | Secretion3-152 . | Secretion/translation . |
|---|---|---|---|---|---|
| Normal | 1.0 ± 0.1 | 1.0 ± 0.3 | 1.0 ± 0.2 | 1.0 ± 0.1 | 1.0 ± 0.1 |
| Ser2119Tyr | 0.7 ± 0.2 | 0.9 ± 0.5 | 0.9 ± 0.7 | 1.3 ± 0.2 | 1.6 ± 0.6 |
| Ile2098Ser | 0.7 ± 0.12 | 0.7 ± 0.3 | 0.7 ± 0.3 | 0.4 ± 0.1 | 0.6 ± 0.2 |
| Arg2150His | 0.7 ± 0.1 | 0.9 ± 0.2 | 0.9 ± 0.3 | 0.14 ± 0.03 | 0.16 ± 0.06 |
| fVIII Gene . | mRNA3-150 . | Translation3-151 . | Translation/mRNA . | Secretion3-152 . | Secretion/translation . |
|---|---|---|---|---|---|
| Normal | 1.0 ± 0.1 | 1.0 ± 0.3 | 1.0 ± 0.2 | 1.0 ± 0.1 | 1.0 ± 0.1 |
| Ser2119Tyr | 0.7 ± 0.2 | 0.9 ± 0.5 | 0.9 ± 0.7 | 1.3 ± 0.2 | 1.6 ± 0.6 |
| Ile2098Ser | 0.7 ± 0.12 | 0.7 ± 0.3 | 0.7 ± 0.3 | 0.4 ± 0.1 | 0.6 ± 0.2 |
| Arg2150His | 0.7 ± 0.1 | 0.9 ± 0.2 | 0.9 ± 0.3 | 0.14 ± 0.03 | 0.16 ± 0.06 |
Factor VIII (fVIII) and hypoxanthine guanine phosphoribosyl transferase (HPRT) messenger RNA (mRNA) steady-state levels were determined by quantitative real-time PCR of total mRNA extracted from Chinese hamster ovary (CHO) cells 72 hours after transfection with an expression vector for mutant or normal fVIII. After correction for HPRT levels, fVIII mRNA levels were expressed relative to fVIII mRNA levels in CHO cells transfected with the expression vector for normal fVIII.
fVIII translation efficiency was determined by a short [35S]methionine labeling of CHO cells 72 hours after transfection with expression vector for mutant or normal fVIII. Total cell lysates were analyzed by immunoprecipitation with monoclonal antibodies directed toward fVIII light and heavy chains, followed by SDS-PAGE and autoradiography. Results are expressed relative to the levels of fVIII single-chain primary translation product in CHO cells transiently transfected with the expression vector for normal fVIII.
fVIII secretion was assessed by measuring fVIII:c activity accumulated in conditioned medium of CHO cells during 12 hours preceding [35S]methionine labeling or RNA extraction. Results are expressed relative to fVIII:c activity in the conditioned medium of CHO cells transfected with an expression vector for normal fVIII. Mean ± SD of 2 experiments are shown.