EMBRYONIC STEM CELL SCREENING
CFCM guarantees the picking of 144 clones. In case a higher number of resistant clones are present, they will be picked until a maximum of 384 clones.
Clones are picked in 96-well plates, grown and expanded. For each plate will be done 2 replicas of cells grown onto MEF that will be stored at –80°C refrigerator and 2 replicas of cells grown onto gelatin that CFCM will use to extract DNA.
The screening will be performed by Southern blot. This is the most reproducible technique to screen for recombinant clones.
Before to start the electroporation:
- The User should test the probe to use during the screening on wild type Sv129 DNA provided by the facility and cut with a specific Restriction Enzyme (RE). The choice of the probe and RE is critical to discriminate only recombinant and not random insertions. Here below an example of how to choose the probe to test the correct integration of the 5’ arm.
You should digest your DNA with a restriction enzyme (RE) that cut outside the 5’ homologous region inserted into the gene-targeting vector. The probe should be designed between the sequences contained between the RE used to cut DNA and the 5’ homologous region. This probe shows you the wt allele and a longer recombinant band. The same should be done at 3’. Using both probes the intensity of wt and recombinant band should be the same.
- CFCM will reproduce the hybridization conditions identified by the User on wild type DNA. If conditions are reproducible, CFCM electroporates the gene targeting construct.
- CFCM performs the Southern blot analysis of clones grown on 96-well plates using one of the two probe. Positive clones will be confirmed on the replica using the other probe.
- CFCM thaws and expands positive clones. Before injection CFCM retests expanded clones by Southern blot. We can give recombinant DNA to the User in the case want to test by itself clones. In the meantime, clones are tested for the absence of mycoplasm and for the correct number of chromosomes.
WHAT WE NEED:
- The User should provide 400 ng of probe to screen 5’ arm integration and 400 ng of probe to detect the correct 3’ integration
- A picture of the agarose gel of two probes
- A map of the construct and a map of the targeting strategy with the indication of REs to use to cut DNA for the screening
- Indications of the Southern conditions used to test probes (such as hybridization temperature and washing conditions)
Here below the scheme of rules and time required for each step of your project.
|Home||Guidelines||Pronuclear inj.||Lentiviral inf.||Blastocyst inj.|
|Rederivation||Electroporation||ES screen||Lentiviral Prep||Costs|