Stable cell line construction:
You choose a constitutive or inducible* promoter, a tag,
a selection marker and a fluorescent marker (see vector map schemes).
Either you provide target templates or we design, synthesize or obtain it from a cDNA collection.
The service AMSBIO provide:
- Clone your gene or shRNA into our lentiviral vector.
- Generate lentivirus.
- Transduce the cell line of interest.
- Select the stably transduced, high expression cells.
- Validate the genomic integration of the target by genomic PCR.
- Validate the high-expression clone by Western Blot (if applicable).
- Produce two cryogenic preserved vials of stable cells and a final report.
Why use AMSBIO stable cell line services :
- Safe-to-use (self-inactivating) lentiviral particles can deliver your gene into a wide range of cell lines including
non-dividing, primary or stem cells.
- Engineered in-house lentiviral vector for highly efficient gene integration into cell genome.
- Our lentivectors have the strongest promoter for the highest protein expression in mammalian cells.
- You can choose to have an inducible* or constitutive promoter.
- Our experts have years of experience in lentiviral cloning and expression.
- Fast turnaround time.
- The best price and the best quality in its class.
Some of AMSBIO's other services :
High titer ready-to-use shRNA Lentivirus
for your targets (around 107
Generation of stable cell lines in almost any mammalian cell line.
Protein expression in mammalian suspension cells using lentiviral transduction.
Affinity purification (His-tag) of recombinant proteins expressed in mammalian cells.
*Additional information on our inducible suCMV promoter :
Without further intervention, our inducible lentiviral particles drive regular constitutive expression.
To take advantage of the promoter’s inducible properties, a repressor protein TetR must be present. The expression of TetR can be achieved by using our
pre-made Tet-repressor lentiviral particles
or by using our stable Tet-repressor cell line.
What are the advantages of using lentivirus to generate stable cell lines?:
Initially, the expression of the target protein is repressed by TetR, but addition of tetracycline induces expression in a dose dependant manner.
To make a cell line with stable, constitutive expression of a target gene, the target DNA has to be integrated into the host cells' genome.
With random integration (through processes such as transfection), a large variety of expression is dependent on the transcription levels at the integration sites.
Random integration often results in the independent integration between the target and the selection marker,
which requires a considerable amount of screening work for selecting positive clones that are resistant and show a high expression level.
In general, less than 10% resistant clones express the transgene.
In contrast, lentivirus transduction has a tendency to integrate in high transcription level sites (hot-spots) with a full virus genome.
An engineered lentiviral transfer vector with an embedded matrix-attachment region (MARs) sequence may provide position-independent transgene expressions.
Compared to conventionally stable cell line construction, lentivirus has a much higher positive clone rate
and the target always co-integrates together with the selection marker,
thus substantially reducing the cost, labour and time spent selecting high-level expression stable clones.