Production of Transgenic Mice or Rats

via Pronuclear Microinjection

•  Introduction

The introduction of foreign DNA (transgenes) into the genome of laboratory animals allows studies of gene expression and protein function within live animals. The mouse is the species of choice for most investigators due to their relatively low cost and the extensive research that has been done in murine models in the past resulting in increased knowledge of their genetics and physiology in various strains.   In some cases, however, investigators prefer to use rats due to factors such as physiological and pathological similarities to humans or increased size.   The TAF offers the opportunity to produce transgenic mice and rats.

Essentially the steps necessary for the production of transgenic mice/rats are:

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 free from prokaryotic vector sequences
3. purification of the transgene for microinjection
4. microinjection of the transgene into the pronuclei of fertilized ova to generate mice/rats containing copies of that transgene integrated randomly in their genome

•  Getting Started

Prior to the start of pronuclear microinjections, the investigator will need to meet with the TAF staff.   This consultation will enable us to determine the specific needs of the investigator, review the design of the transgenic vector, and discuss fees for various services. Please contact Anne Griep (aegriep@wisc.edu) or (608) 262-8988 to schedule an initial meeting.

•  Guidelines for Transgene Vector Design

We recommend incorporating the following features in your transgenic vector.

1. promoter with a known cellular expression profile
2. a genomic clone or cDNA and intron fragment containing splice donor and receptor sites
3. a fragment containing poly adenylation addition sequences
4. well characterized restriction sites that will allow the isolation of a transgenic fragment free from prokaryotic vector sequences

•  Purification of Transgene for Microinjection

Highly purified DNA is essential for successful transgenic production. Particles within the DNA could easily clog the microinjection needle and/or interfere with integration of the DNA.   Therefore, we recommend that you isolate the fragment by electrophoresis followed by electroelution.  DNA can be purified by the investigator or by TAF for a time and material recovery fee.

TAF also has experience microinjecting BAC and PAC DNA.   However, isolation methods are different.   We recommend using the Nucleobond column by Clontech .

•  Microinjection of DNA into Oocytes

Checklist:

1. complete a billing request form
2. provide an IACUC approved animal protocol number
3. provide DNA along with a gel picture showing one distinct band and concentration.   At this point, the transgene is put in line for next available injection dates.   TAF will verify the fragment size and dilute the DNA for microinjection 
4. provide a map of your transgene and vector

•  Transgenic Mice

TAF routinely microinjects transgenes into the pronucleus of FVB/n and C57BL/6 1-cell embryos. Other strains will be considered only after meeting with TAF staff to discuss the project. The investigator may incur additional costs for the use of other strains as embryo donors if costs of materials and time required to complete the project are greater than for our standard strains.

Following the microinjections, the embryos are transferred into the oviducts of pseudopregnant recipients. Pups should be born 19 days later and weaned 3 weeks after birth. Investigators are provided with tail DNA samples from each potential founder and have three weeks to complete genotyping the mice.   We recommend Southern Blot analysis as the preferred method of genotyping. If analysis takes longer than three weeks, daily animal per diems will be charged to the investigator. The investigator must provide a copy of the genotyping results to the TAF staff. The TAF then will breed up to three 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 invstigator when the progeny are three weeks old. Any remaining founders will be shipped without breeding to the investigator. Mice are transferred to investigator only after an animal transfer form has been completely by both the investigator and veterinarian staff from the relevant school.

A contract is considered complete when 20-25 weanlings have been produced. Half-contracts are available upon request and fees prorated accordingly.

•  Transgenic Rats

TAF routinely microinjects transgenes into the pronucleus of Sprague-Dawley (outbred) 1-cell embryos and has also successfully produced transgenics on an F344 (inbred) background. Other strains will be considered only after meeting with TAF staff to discuss project.   The investigator may incur additional costs for the use of other strains as embryo donors if costs of materials and time required to complete the project are greater than for our standard strains.   Following the microinjections, the embryos are transferred into the oviducts of pseudopregnant recipients. Pups should be born 22 days later and weaned 3 weeks after birth.   For further details regarding microinjection or embryo transfer protocols, click on this link.

At weaning, investigators are provided with tail DNA samples from potential founders and have three weeks to complete the genotyping.   We recommend Southern blot analysis as the preferred method of genotyping.   If analysis takes longer than three weeks, daily animal per diems will be charged to the investigator.   The investigator must provide a copy of the genotyping results to TAF staff.   We will then breed the first three founders when they reach breeding age to wild-type mates to produce one F1 litter from each founder.   Any remaining founders will be shipped to the investigator for breeding. Rats are transferred to the investigator only after an animal transfer form has been completed by both the investigator and veterinarian staff from the relevant school.   A contract is considered completed when 35-40 weanlings have been produced.