• 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).

User guide:

1. TKTC provides vector plasmid, you make your own construct or we make the construct with a fee.
2. TKTC purifies the provided DNA construct then microinjects it into mouse pronucleus with PhiC31 integrase mRNA.
3. 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.
4. TKTC will genotype pups to confirm insertion by universal PCR primers. You will do further genotyping to verify the specific gene integration.
5. If requested, TKTC will further confirm insertion by specific PCR and DNA sequencing.
6. Here is genotyping info and strategy.
7. For external user, we will provide genotyping to confirm specific insertion. 

How to request: 

1. Log-in iLab, select Transgenic Services, then Site-specific Knock-in transgenic mice.
2. Fill in request form, attach construct plasmid map with DNA sequences (or send to us by email).
3. Submit request. Get vector plasmid DNA from TKTC, and clone your gene in between 2 attB sites.
4. Get vector plasmid DNA from TKTC, and clone your gene in between 2 attB sites.
5. Provide TKTC 20ug construct plasmid DNA with map and DNA sequences.
6. TKTC will update you injection dates,  genotyping results, and mouse transfer notification.