Transgenic mice were generated using recombinase-mediated integration in embryonic stem cells or zinc-finger nuclease-aided genomic targeting combined with miR30-shRNA technology.
If these novel mutant lox sites could also improve integration efficiency in ES cells, they would be useful tools for Cre-mediated integration in mammalian genomes.
This PCR product was subsequently cloned in the pABC plasmid with the use of restriction-ligation with KpnI (Choi et al. 2009); pABC harbors two attB sites that allow for PhiC31-mediated integration in the attP sites of a MiMIC element.
First, the specificity and efficiency of Cre recombinase mediated integration into pM24-BAC (Step1) was examined.
The I-SceI enzyme mediated integration, or SEMI, in combination with a Δtku70 mutant has a synergistic effect on homologous recombination efficiencies as 90 100% of the transformants exhibited integration of the expression cassette at the homologous site.
Here, we analyzed the features of ϕC31-mediated integration into pseudo attP sites in the human genome.
tbrd-1Δc was subsequently inserted into the transformation vector pUAST containing the attR cassettes for Gateway® recombination cloning technology (InvitrogenTM), the attB recognition site for phiC31 mediated integration at attP destination sites in the genome as well as the coding sequence for the C-terminal tag eGFP (pUAST-attB-rfa-eGFP; kindly provided by S. Bogdan, Münster; unpublished).
The construct was linearized using KpnI and used in restriction enzyme mediated integration (REMI) transgenesis (Kroll and Amaya, 1996) using purified sperm nuclei and unfertilized eggs from wildtype frogs purchased from Nasco Biology (Fort Atkinson, WI).
Restriction enzyme mediated integration (REMI) mutagenesis was used to create insertional mutations in an ampA overexpressing (ampAOE) cell line.
To determine further if φC31 integrase mediated integration of plasmid DNA into the cells that generated tumors, we tested all dissected tumors for integration into the dominant pseudo attP site in the mouse liver genome known as mpsL1 [9], using a nested PCR.
