When should I consider reversible integration (Flp-In system) vs irreversible integration (Jump-In system)?
Use irreversible integration (Jump-In system) if the transgene should be sustained in the mammalian genome for a long time. Use reversible integration such as Flp-In system if the transgene needs to be replaced with another gene of interest after a short period of time.
What controls do I need in the Jump-In system to check for the successful retargeting of the platform line?
The second step in targeted integration is the retargeting event mediated by the R4 integrase where the genetic elements of interest are site-specifically transferred from the retargeting expression construct (created using the MultiSite Gateway Pro module) onto the genome of the platform line. This integration event also positions the EF1alpha promoter upstream of the blasticidin, neomycin, or eosin resistance gene (i.e., "promoterless" selection marker), thus allowing the selection of transformants that are successfully "retargeted" using the appropriate selection agent. Although you select from successfully retargeted clones using the blasticidin, Geneticin, or Zeocin antibiotic, you may also perform a nested PCR to amplify the region from the EF1alpha promoter to the appropriate resistance gene. You can amplify the hygromycin resistance gene as a positive control. Similar to the platform line creation, you may also perform a Southern blot analysis with a probe designed for your gene of interest.
What controls do I need in the Jump-In system to check for the presence of the R4 target site after the creation of the platform cell line?
A platform cell line is created when the R4 attP retargeting sequences are site-specifically inserted into the mammalian genome via PhiC31 Int-mediated recombination. In addition to the R4 retargeting sequences, this integration event introduces the hygromycin resistance gene under the control of the HSV TK promoter and the promoterless Bsd, Neo, or Zeo resistance marker, depending on the platform vector used (i.e., pJTI/Bsd, pJTI/Neo, or pJTI/Zeo). Although you select for transformants carrying the R4 retargeting sequences by their resistance to hygromycin, you may perform PCR analysis to check the integrity of the R4 attP retargeting sequences. For this, we recommend amplifying the region from the R4 attP sequence to the appropriate resistance marker (depending on the platform line used) using the genomic DNA from the platform line. A nested PCR is recommended to reduce the high background you may observe with only primary PCR. Alternatively, you may create a labeled DNA probe by PCR amplifying an approximately 1.5 kb region covering the retargeting sequences, and then perform a Southern blot analysis. The Southern blot will also act as an additional check to verify that only a single copy of the retargeting sequence is integrated into the genome.
How much DNA or what controls do I need to include in the Jump-In system in order to get one integration event?
The amount of DNA to be used to obtain single copies should be determined by control experiments done in the absence of integrase. The same amount of DNA that yields less than 5 colonies in the absence of integrase should be used in the presence of integrase. Typically, the integrase expression plasmid makes up most of the amount of DNA used for transfection.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
When should I use the Jump-In Fast system vs the Jump-In-TI (Targeted Integration) system?
Use the Jump-In Fast system if you need stable mammalian expression and want to quickly generate well-expressing clones. You can have well-expressing clones with one or more integrations at the PhiC31 pseudo-att P sites. A Southern blot is necessary to confirm the number of integrated events.
Use the Jump-In TI system if you need isogenic expression, where every cloned gene would be expressed from the same locus in the same background with no chromosomal position effects.
