High‐level recombinant protein production in CHO cells using lentiviral vectors and the cumate gene‐switch
Bibliographic record
Abstract
Fast and efficient production of recombinant proteins for structural and functional studies is a crucial issue for research and for industry. To this end, we have developed an efficient system to generate in less than 2 months, starting from the cDNA, pools of CHO cells stably expressing high-level of recombinant proteins. It is based on lentiviral vectors (LVs) for stable transduction coupled with the cumate gene-switch for inducible and efficient gene expression. Transcription is initiated upon binding of the cumate transactivator (cTA) or the reverse cTA (rcTA) to the CR5 promoter. Binding of cTA or rcTA is prevented or induced by addition of cumate respectively. We first validated the CHO/LV production system with an LV carrying the secreted alkaline phosphatase (SEAP), whose expression was linked to the green fluorescent protein (GFP) through an internal ribosome entry site (IRES). CHO cells stably expressing the cTA (CHO-cTA) were transduced at various multiplicity of infection (MOI). Pools of cells were incubated at 37 and 30 degrees C during 10 days. Optimal SEAP production (65 microg/mL) was achieved at 30 degrees C with a MOI of 200. The pool stability was demonstrated for 48 days of culture by GFP expression analysis. The system was also evaluated using LV expressing three typical therapeutic proteins (a protein made up of the extracellular domain of CD200 fused to IgG Fc region [CD200Fc], a chimeric antibody [chB43], and erythropoietin [EPO]). CHO cells expressing rcTA (CHO-Cum2) were transduced with these LVs at a MOI of 200 and production was tested at 30 degrees C. After 13 days of culture, 235, 160, and 206 microg/mL of CD200Fc, chB43, and EPO were produced, respectively. The ON/OFF ratio of these pools was equal to 6 for CD200Fc, 16 for chB43, and 74 for EPO. In conclusion, this system should be very useful to produce mg quantities of recombinant proteins in a timely manner in serum free suspension culture of CHO cells for preclinical studies.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".