The workflow of stable cell line establishment is shown in Figure 1. An effective way to increase mammalian system productivity is to amplify the copy of YFG (Your Favorite Gene) in the host cell. Creative BioMart offers MTX/DHFR pressure selection strategy to facilitate YFG amplification, helping our CHO cell users obtain high productivity positive cell clone.
Figure 1. Workflow of Stable Cell Line Establishment
Chinese hamster ovary (CHO) cells are the most widely used cells in bioengineering. It had participated in the production of more than 70% recombinant biopharmaceutical proteins. CHO cells are immortal and can be passaged for hundreds of generations.
At present, the most commonly used pressure selection system is the dihydrofolate reductase system (DHFR). The system is based on DHFR deficient CHO cells. After the plasmid harboring the DHFR gene is transfected into the host cell, cell clones growing in the selected medium could be obtained. DHFR could be inhibited by methotrexate (MTX). Adding MTX to the culture medium and increasing the concentration gradually will force the cell clone with a non or low copy of DHFR gene to die. Only clones that contain high DHFR copy can survive.
Dihydrofolate reductase is a co-amplified gene. With the forced amplification of DHFR gene, the YFG linked with it will be amplified hundreds to thousands of times.
| Service Name | Service Description | Timeline |
| Gene and Vector preparation |
|
2-4 weeks |
| Stable Cell Pool Screening |
|
6-8 weeks |
| MTX Amplification and Subclone |
|
2-3 months |
| Fed-batch Culture |
|
3-4 weeks |
| Cell Line Stability Evaluation |
|
2 weeks |
| Research Cell Bank Preparation |
|
2 weeks |
| Scale-up Protein or rAb Production |
|
Variable based on request |
Two strategies regarding transfection are currently available at Creative BioMart.
Strategy 1——YFG linked to a DHFR minigene on a single plasmid.
Strategy 2—— YFG and DHFR minigene on separate plasmids.
The plasmid carrying the DHFR gene and the expression plasmid carrying YFG are co-transfected into CHO-DHFR cells, this method allows a high ratio of YFG to DHFR. Co-amplification is not assured but commonly occurs.
Creative BioMart's MTX Amplification Protocol
Customers who need our assistance are more than welcome to contact us or submit an online inquiry. Our professional team will provide you with detailed guidance and solution ASAP. If you have any special needs or are interested in learning more about Creative BioMart's protein expression services, please feel free to inquire about more details.
