Production of Transgenic Mice or Rats via Pronuclear Microinjection

Create your Transgenic Mouse or Rat in four easy steps:

  1. construction of a transgenic vector containing a suitable promoter, a genomic clone or DNA and intron fragment, and polyadenylation addition sequences 
  2. isolation of the transgenic fragment from prokaryotic vector/backbone sequences 
  3. purification of the transgene for microinjection 
  4. microinjection of the transgene into the pronuclei of fertilized ova to generate mice/rats containing random copies of that transgene in their genome

Please contact Kathy Krentz (kjkrentz@wisc.edu) to schedule an initial meeting.

      1. Guidelines for Transgene Vector Design

We recommend incorporating the following features in your transgenic vector. We commonly use SGI-DNA for all synthesis of plasmids.
       a. promoter with a known cellular expression profile
      b. a genomic clone or cDNA and intron fragment containing splice donor and receptor sites
      c. a fragment containing polyadenylation addition sequences
      d. well characterized restriction sites that will allow the isolation of a transgenic fragment free
         from prokaryotic vector sequences

     2. Purification of Transgene for Microinjection

Highly purified DNA is essential for successful transgenic production. Our facility will purify your digested DNA. Bring 30-50 µg digested DNA to our lab with a gel picture clearly indicating which band we need to purify. 

TAF also has experience microinjecting BAC and PAC DNA.   However, isolation methods are different.   Please contact us for the latest isolation procedure. 

    3. Microinjection of DNA into Oocytes

TAF routinely microinjects transgenes into the pronucleus of C57BL/6J one-cell mouse embryos or Sprague Dawley rat embryos. TAF has experience using other mouse strains such as FVB, NOD, NOD/Shiltj, ICR/HaJ, 129S1, and Btbr and rat strains F344, ACI, Lewis.  Transgenics can be made in these strains but investigators must purchase the wild-type animals. Other strains will also be considered. 

Following the microinjections, the embryos are transferred into pseudopregnant recipients and pups born. At weaning, animals are ear-tagged and samples taken. Investigators are provided with tail DNA samples from each potential founder. The investigator must provide a copy of the genotyping results to the TAF staff. The TAF then will breed up to two founders when they reach breeding age to wild-type mates to produce one F 1 litter from each founder. The founder and litter are transferred to the investigator when the progeny are three weeks old. Any remaining founders will be shipped without breeding to the investigator.