DNA Microinjection

Direct microinjection of DNA into the male pronucleus of a mouse zygote has been until recently the method most extensively used in the production of transgenic mice. If the foreign DNA integrates into the mouse chromosomal DNA at the one-cell stage, the animal will contain the injected DNA in every cell, including those of the germ line. If, however, integration occurs later, the resulting animals will be mosaic for the presence of the injected DNA. The number of copies of integrated DNA can vary from one to several hundred, arranged primarily in tandem head to tail arrays.

What the Facility will Provide

Upon receipt of the DNA construct, proven by documentation to be of high quality and suitable for microinjection (see below), we will proceed to inject a minimum of 150 fertilized eggs. After overnight incubation, normal 2-cell embryos will be transferred into foster females and their pregnancy will be monitored (the gestation period of the mouse is 19-20 days). Two weeks after birth of potential transgenic pups, the project scientist will be notified of the number of pups. The facility will carry out tail clipping, animal tagging and genotyping by PCR according to the conditions set by the project scientist. At weaning, the positive founders will be transferred to the care of the individual investigators as per ULAR procedures. If these studies (with adequate controls) fail to demonstrate the existence of at least one transgenic animal, we will re-inject at least 80 eggs at the cost of additional animals used to the investigator. If, however, no transgenic animal is detected after the second trial, a meeting will be held between the scientist, the Facility Director, and the Technical Director to discuss the DNA construct, the tail biopsy analysis, and further plans to be agreed upon. On average, 230 injected eggs should yield at least one (and usually more) transgenic mouse.

It is well understood that many factors can affect the production efficiency of transgenic mice. Such factors include:

Number of eggs surviving the injection and developing to 2-cell stage; this will determine the number of transfers into foster females. It is important to have a DNA preparation pure of any contaminant and at the appropriate concentration so that toxic effects to the eggs can be avoided.
Successful pregnancies: although every transfer promises a certain number of pups, that number may vary greatly due to embryo death in utero. This lethality may in certain settings be related to the type of DNA construct used.

Transient transgenics

For investigators who wish to examine DNA integration and expression in founder transgenic embryos, we can provide similar DNA injection schedule for a reduced fee. We will inject 150 eggs, transfer the developing embryos into foster females, and arrange with the project scientist to receive the pregnant females on the assigned gestational date. We would still need to receive a report on the number of embryos harvested from each female and the frequency of transgenics among them to monitor our quality control.

Investigator’s Responsibilities

In general terms, the investigator must prepare the DNA to be injected and assume responsibility for screening and care of the weaned animals. Specifically, in order to provide you with this service, it is requested that the investigator:

Contact Jean Richa (215-573-3023) to discuss the DNA construct and the schedule of injections.
Provide the facility with a high-quality DNA preparation of your construct with documentation of its purity and an estimate of its concentration (The recommended protocol for DNA preparation can be found in the “Protocols” page.)
Complete a service request Online at: http://www.med.upenn.edu/apps/my/transcore/user, and submit evidence of approval of the IACUC and IBC committees for the project.
The investigator can have the core screen the tail biopsies at no added cost by providing PCR primers as well as negative and positive control samples.
Undertake the care of the potential transgenic pups submitted to the project scientist. These mice can no longer reenter the barrier colony. It is the responsibility of the individual investigator to arrange for animal housing.

Please also consult additional guidelines related to this service under the “Protocols” section of this site.