Conventional transgenes by pronuclear injection

Pronuclear injection (PNI) is the traditional method to generate transgenic founder mice. The transgene randomly integrates into the host genome. Beware that each founder line is different and needs separate characterisation. Also, chromosome shattering at the integration site can lead to phenotypes unrelated to the transgene. A better method in most cases may be the integration into safe harbours like ROSA26, TIGRE, and H11. The randomness of conventional transgenesis can, however, be used to your favour if you want to screen for particular expression pattern and strength.

This service offers generation of transgenic founder animals by pronuclear injection. Injections are routinely performed in C57BL6/J background, with other backgrounds possible on request (price may vary). The embryo transfers following microinjections are performed under SPF conditions, thus allowing founders to be introduced into most facilities. We routinely obtain 3-5 transgenic founders for each project. Please contact us directly for special requests such as injections in other mouse strains or BAC injection.

1. A completely filled out request form
2. The GMO datasheet as requested by the veterinary office
3. An iRATS breeding project opened under researchers own license to be used for weaned offspring (iRATS users only)
4. 10 μg of DNA at a concentration of >70ng/μl. (Note: The transgene should be cut out such that bacterial vector sequences are   removed since they can induce CpG methylation and thus transgene inactivation. Purification from agarose gel should be performed with Kits such as QiaExII (Qiagen). DNA should be free of endotoxin.)
5. The genebank (*.gbk or *.gb) file of the annotated DNA sequence
6. A validated genotyping PCR approach (including gel pictures), tested with WT genomic DNA spiked with the linearized transgene at various concentrations

The cost of this service is 7300 CHF for inbred (C57BL/6J) mouse strains. This fee covers the donor animals, the injection of DNA, embryo transfers, collection of biopsies, hygiene check and subsequent animal care costs until the founders are turned over to the researcher. An additional fee of 700 CHF will be charged when founders should already be bred to wt animals, before transfer to the target facility.

We normally try to process your request as soon as they arrive. In case of a backlog of requests, the date of DNA receipt and the number of requests submitted by individual groups will determine the sequence in which the projects are carried out. In case of two successful injection rounds that do not yield transgene positive founders, we inject a linearized GFP construct in some of the embryos as internal control in the third round. If we detect GFP positive founder animals among the offspring we consider the targeting construct toxic and charge the regular price. 

For terms and conditions please click  here.

In case of questions, please contact Johannes vom Berg before submitting a microinjection request. Every Friday we offer a “Targeting Club” at 9:00 am in our seminar rooms (Wagistr. 12, Schlieren or Sternwartstrasse 6, Unispital, Zürich), where you are welcome to discuss your project with us (see also upcoming dates).