Transgenic Mouse Services

Integration of genes into the mouse genome can serve as an experimental system to study normal as well as altered gene expression.  Such studies can also serve to identify genes essential for normal development and generate animal models for human diseases.  Direct microinjection of DNA into the male pronucleus of a mouse zygote has been 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 transgenic animal will contain the injected DNA in every cell, including those of the germ line.  The number of copies of integrated DNA can vary from one to several hundred, arranged primarily in tandem head to tail array. The Core offers a number of services to assist our investigators in the generation of transgenic mice. While the design of transgene DNA is not offered in-house, we can highly recommend trusted partners that can perform all DNA design and construction steps at relatively low cost to our investigators. Services offered by the Core include design and/or execution of a PCR-based screening strategy, DNA injection into fertilized oocytes and tail DNA preparation for PCR-based screening, along with the care and housing of the mice up to the stage where they would be transferred to the participating investigator. Investigators can chose to have transgenic mice produced either on a hybrid (C3HxC57BL/6) or on a pure C57BL/6 background (application form).

DNA Injection: Upon receipt of the completed application form and the DNA construct, proven by documentation to be of high quality and suitable for microinjection, the core will proceed to clean the DNA sample and to inject a minimum of 300 fertilized eggs (usually enough to generate 2-10 individual transgenic founder mice).  Embryos will be transferred into foster females and their pregnancy will be monitored (the gestation period of the mouse is 19-20 days).  Ten days after birth of potential transgenic pups, the Core will carry out tail clipping and animal tagging.  Tail biopsies will be conveyed to the investigator for analysis by Southern blotting, PCR, or both (unless the investigator has previously arranged for the Core to prepare tail DNA and perform a PCR-based screen for an additional fee).  At this point, the core will ask the investigator to process the samples as quickly as possible (in two weeks).  If no result is received, there will be $2 charge per cage per day.  After weaning, the tagged pups will be transferred to the care of the individual investigator who will be responsible for subsequent maintenance. Since cage costs are high and space is limited, investigators should make certain that screening procedures are working prior to initiating transgenic work with the core.  Investigators must report the results of screening with documentation (gel photo, etc.) within 14 days.  This is necessary for core personnel to maintain and improve their efficiency.  If these studies (with adequate controls) fail to demonstrate the existence of at least one transgenic animal, we will re-inject at least 200 eggs at no additional cost to the investigator.  If, however, no transgenic animal is detected after the second trial, a meeting will be held between the scientist, the Core Director, and the Core personnel to discuss the DNA construct, the tail biopsy analysis, and further plans to be agreed upon. It is well understood that many factors can affect transgenic mouse production efficiency, including:

• 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.  Therefore we will clean the DNA samples after receiving it.

• 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 be closely associated with the type of DNA construct used.