CB-Gene's Question 1: It is generally believed that during this construction step, the FRT will integrate into the cell's transcriptionally active region. This means that for subsequent expression of the recombinant exogenous target gene, this integration step provides a certain degree of assurance for overexpression of the exogenous protein.

 

For specific integration locations, please consult our ISS (Integration Site Search) service.

CB-Gene's Question 2: In a conventional plasmid transfection protocol, how many copies can be integrated into the host cell? What is the optimal number of copies? Generally speaking, when constructing Flp-FRT mother cells, integration occurs randomly after transfection, meaning that multiple copies will be integrated into different chromosomal locations in the host cell. The more copies integrated, the more likely the target gene will be recombined and integrated, indicating higher expression. However, when recombining the target gene, the integration efficiency of multiple copies may not be 100%, resulting in mixed clones (inconsistent expression levels between clones). Sometimes, for target proteins that do not require high expression, Kebai recommends selecting clones with a single integrated copy. This way, any clone that passes resistance screening is, in principle, a single clone, eliminating the need for limiting dilution cloning. 

 

 

CB-Gene's internal spot Flp-FRT cells

 

2. Integration of the target gene:

An expression vector containing the target gene and an expression vector containing Flp recombinase are co-transfected into the cells from step 1. Flp recombinase mediates site-specific recombination between the FRT sites in the host genome and those in the expression vector, achieving integration of the target gene and enabling stable expression under the control of the promoter.

 

Carrier 1:

Overexpression of Flp recombinase, no resistance required, no stable integration required

 

 

Carrier 2:

Contains target gene and second resistance gene selection marker

 

 

Reorganization process：

 

 

Final formation：

After recombination, positive cells can be selected through the second resistance gene, and the cells are tolerant to resistance gene-2 and sensitive to resistance gene-1.