Note - we now also offer targeted transgenesis at the H11 locus mediated via microinjection of Cas9 RNP and plasmid targeting vectors in zygotes.
This service is designed to insert single-copy transgenes via homologous recombination into the ROSA26 locus. Unlike the traditional method of injecting transgene DNA into the pronuclei of fertilized eggs, where integration occurs at random locations with variable copy numbers and unpredictable expression levels, this service results in consistent levels of ubiquitous expression driven by the endogenous ROSA26 promoter. If the client wishes to control expression with an exogenous promoter, insulator elements can be inserted in the targeting vector.
This service offers the following advantages over the traditional method of pronuclear injection:
- Similar levels of expression in all founder lines.
- A known locus of integration.
- Transgenes are always inserted as a single copy, minimizing the chance that they will be silenced by methylation or lost via recombination.
- As the site of integraton is known, only a single line of "transgenic" mice is required.
How it works:
Cloning vectors are available from Addgene containing homology arms that are proven to result in a high rate of homologous recombination (see Fig. 1). We recommend cloning the transgene into pBigT, which contains a floxed neo cassette, then excising with PacI and AscI and subcloning into pROSA26-PA, which contains the homology arms. The client is responsible for all cloning steps, checking for cloning errors, and supplying at least 50 ug of plasmid DNA purified with the Qiagen Endo-Free Plasmid Maxi kit. The TMF will linearize the construct and perform the final purification prior to electroporating it into ES cells. For clients who prefer to use Gateway cloning technology, Addgene offers a Gateway-ready version of pROSA26-PA, called pROSA26-DEST.
The TMF will grow the ES cells under selection, pick a small number of resistant colonies and screen them by Southern blot, using a probe for the shorter of the two homology arms. Based on our experience so far, around 50% of the colonies should have the transgene correctly integrated. Using the standard homology arms in pROSA26-PA allows us to use a proven probe for all Southern blots.
The client should then request at least 2 correctly targeted colonies to be expanded by us and cryopreserved in multiple vials stored in liquid nitrogen in the TMF. We also strongly recommend our clients to have us count the chromosomes in 20 metaphase spreads of each clone that they want us to inject. Clones with less than 50% euploid spreads are unlikely to produce chimeras. Clone expansion and chromosome counting will incur additional fees. Injection into blastocysts to produce chimeric mice would then be performed under our standard ES cell injection service. Resulting chimeras can be bred by the client (or by the TMF under our standard breeding service) to produce heterozygous founder mice.
Fig. 1: Southern blot results for 40 colonies after targeting the ROSA26 locus. The wildtype locus produced a band of 5.8 kb in all colonies, and 27 colonies (68%) display an additional band at 3.1 kb, indicating correct targeting with the transgene.
In addition to the Service Request Form, you must supply the following:
- At least 50 micrograms of your targeting construct plasmid, purified using the Qiagen Endo-Free Plasmid Kit, and dissolved in TE.
- A gel picture of uncut plasmid and plasmid cut with the restriction enzyme that we will use to linearize the plasmid.
It is the client’s responsibility to make sure the targeting construct has been engineered correctly, mapped, sequenced, and tested.
The minimum time needed to go from electroporation of the targeting construct to clone DNA ready for genotyping is about 4 weeks. The actual time may be somewhat longer if other targeting jobs are already in progress.
Southern blotting turnaround times can vary, depending on how many projects are in progress. At present, the minumum time is about 10 days.
Clone expansion and DNA extraction require an additional 2 weeks.
We guarantee to produce at least 2 correctly-targeted clones, based on results from the first Southern blot.
The standard Targeted Transgenesis service includes the following:
- Linearization and purification of the targeting construct DNA.
- Electroporation into ES cells.
- Growth of cells under selection.
- Picking at least 24 drug-resistant colonies into a 96-well plate.
- Splitting the colonies into duplicate plates.
- Freezing one plate and extracting DNA from the other plate.
- Digestion of ES cell colony DNA with restriction enzyme.
- Agarose gel electrophoresis and gel photography.
- Capillary transfer of digested DNAs to nylon membrane.
- Hybridization of membrane with radioactive probe (standard ROSA26 probe supplied by the TMF).
- Washing blot and imaging on phosphorimager.
- Analysis and annotation of image.