Integrase Mediated Transgenesis
- Integrase mediated transgenesis (IMT) was developed in Stanford University by collaboration of Dr. Luo lab and Stanford Transgenic Facility (Tasic, et al, 2011). With this technology, genes can be knocked-in either at the ROSA26 locus or H11 locus. H11 is a novel locus that works equivalently as a commonly used ROSA26 locus (Hippenmeyer, et al, 2010).
- The system is based on that integrase (PhiC31) catalyzes irreversible recombination between attB and attP sites. We developed integrase-mediated site-specific transgenesis in mice via pronuclear microinjection. We use PhiC31 integrase to catalyze recombination between one or two attB sites in a recombinant DNA with three tandem attP sites that were previously inserted into specific loci in the mouse genome (See Figure below).
- Mouse lines with three tandem attP sites that were knocked into the Hipp11 (H11) or Rosa26 loci (Left in Figure) are zygote donor. A mix of dDNA and PhiC31 mRNA was injected into zygote pronucleus. Donor plasmid DNA coded the gene of interest (e.g., GFP) flanked by two attB sites. PhiC31 catalyzes a recombinase-mediated cassette exchange reaction (bottom branch).
- TKTC provides vector plasmid, you make your own construct or we make the construct with a fee.
- TKTC purifies the provided DNA construct then microinjects it into mouse pronucleus with PhiC31 integrase mRNA.
- Please provide cage card stickers to Sim1 G0421 with your APLAC # and PTA. We’ll contact you when the positive mouse is transferred to your mouse room.
- TKTC will genotype pups to confirm insertion by universal PCR primers. You will do further genotyping to verify the specific gene integration.
- If requested, TKTC will further confirm insertion by specific PCR and DNA sequencing.
- Here is genotyping info and strategy.
- For external user, we will provide genotyping to confirm specific insertion.
How to request:
- Log-in iLab, select Transgenic Services, then Site-specific Knock-in transgenic mice.
- Fill in request form, attach construct plasmid map with DNA sequences (or send to us by email).
- Submit request. Get vector plasmid DNA from TKTC, and clone your gene in between 2 attB sites.
- Get vector plasmid DNA from TKTC, and clone your gene in between 2 attB sites.
- Provide TKTC 20ug construct plasmid DNA with map and DNA sequences.
- TKTC will update you injection dates, genotyping results, and mouse transfer notification.