At present, “random integration” is often used to integrate the target gene into the host cell genome when establishing stable cell lines. However, the unpredictable insertion location, copy number and expression level of the foreign gene may all lead to an undesired expression event (so-called position effect). A strategy to overcome the position effect is to integrate the foreign gene into the active transcription region site-specifically by homologous recombination to improve the transcription level of the target gene.
Creative BioMart offers two site-specific recombination systems, Cre / LoxP and Flp / FRT, to help our customers to obtain high productivity positive cell clones.
Technology description
Cre / LoxP recombinant system can mediate the site-specific integration of foreign genes on host chromosomes. Cre recombinase can mediate the precise recombination between a circular plasmid containing a single LoxP sequence and genes or chromosomes containing the same LoxP sequence.
Service content
| Service Content | Service Detail |
| Selection of host cell line | Several different transgenic cell lines containing a LoxP site have been established in advance#1. |
| Gene synthesis & subcloning |
|
| Transfection |
|
| Stable cell line construction |
|
#1: The LoxP sequence can be inserted into the host cell genome through either homologous recombination, transposable element or virus vector.
#2: The Cre recombinase can be added into the system through either co-transfecting on Cre expression vector, Cre mRNA or add pure Cre protein directly.
Technology features
Technology description
Flp recombinase can mediate excision, inversion, integration and translocation between two FRT sites. Similar to Cre / LoxP recombination system, two FRT fragments located on different chromosomes (or plasmids) can be recognized by Flp recombinant enzyme and mediated site-specific integration.
Service content
| Service Content | Service Detail |
| Establish of host cell line | Transgenic cell lines containing an FRT site has been established in advance through homologous recombination#3. |
| Gene synthesis & subcloning |
|
| Transfection |
|
| Stable cell line construction |
|
#3: The FRT sequence can be inserted into the host cell genome through either homologous recombination, transposable element or virus vector.
#4: The Flp recombinase can be added into the system through either co-transfecting on Cre expression vector, Cre mRNA or add pure Cre protein directly.
Technology features
Flp / FRT system has the same advantages as Cre / LoxP. The difference is that the optimal functional temperature of the Flp / FRT system is 30 ℃, so it is more suitable for site-specific integration of mammalian cell lines in vitro.
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.
