Transgenic Research Center In the Cancer Center

Site-specific gene knockin using integrase

We have recently developed a new method to generate site-specific transgenic mice via direct injection into mouse embryos, bypassing ES cell targeting step. Our work was published in PNAS in May 2011 (Tasic, et al, 2011). Genes can be knocked-in either at the ROSA26 locus or H11 locus, a novel locus we identified and works better than the commonly used ROSA26 locus at least for ubiquitous gene expression.

How does our system work?

Our sytem is based on an integrase-mediated method, originally discovered by Dr. Michele Calos, a professor in Genetics Department here at Stanford. Integrases catalyze irreversible recombination between appropriate attB and attP sites. PhiC31 integrase from a Streptomyces phage has previously been used for transgene integration in flies. and low efficiency transgenesis in mice. We developed an integrase-mediated method for sitespecific transgenesis in mice via pronuclear microinjection, with integration efficiencies as high as 40%. We use PhiC31 integrase to catalyze recombination between one or two attB sites in a recombinant DNA with one or more tandem attP sites that we previously inserted into specific loci in the mouse genome (See Figure below).

One or three tandem attP sites were knocked into the Hipp11 (H11) or Rosa26 loci via homologous recombination in ES cells (Left in Figure below). Mice homozygous for one of the modified loci (H11 is shown as an example) served as embryo donors. A mix of DNA and in vitro transcribed PhiC31 mRNA was injected into a single pronucleus of each zygote. The integration of plasmid bacterial backbone (BB) that decreases the transgene expression was avoided either by injecting a minicircle DNA with a single attB site (top branch; in this case, PhiC31 catalyzes a typical integration reaction), or by injecting plasmid DNA where the gene of interest (e.g., GFP) was flanked by two attB sites (bottom branch; in this case, PhiC31 catalyzes a recombinase-mediated cassette exchange reaction). Right, Middle: Green fluorescence of a representative transgenic F0 embryo and the corresponding PCR results that indicate site-specific insertion. The red numbers for the PCR results correspond to the numbers on the H11P3-pCA-GFP transgene scheme below; the same PCR tests can be used for the transgene above. The particular embryo shown was obtained by cassette exchange. Inset: Bright-field image of the same embryo.

How to make a request for this service?

1. Fill out and submit an online requistion form.

2. We will contact you and provide you with an aliquot of an appropriate plasmid vector containing attB site(s).

3. You will make the construct or have us make the contruct for a fee.

4. Provide us with your onstructed plasmid and we will purify it in an RNAase free condition.

5. We will perform microinjection and transfer the pups to your mouse room in about 4 weeks after microinjection.

Strain background

We currently have FVB for H11/attP mice, and mixed background for ROSA26/attP mice. We will have C57BL6 for H11/attP in about 4-6 months.

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