Transgenic Outline: Mice and Rats

 

Thom Saunders with any questions or Meet the Staff.

review of reporter molecules is available. The Core has a nuclear localized lacZ reporter vector (pnlacf) for investigators who wish to characterize regulatory elements in transgenic mice. A completed materials transfer agreement is required before this plasmid can be distributed to investigators. Commercially available vectors that may be useful in transgenic research include: 1) the CMV-IE promoter for widespread gene expression, 2) tetracycline regulated gene expression systems for inducible gene expression, and 3) luciferase and green fluorescent protein reporter genes.

Review Articles) . Under the best circumstances, the transgene is tested for expression in a tissue culture system before transgenic mice are made.

PCR assay to rapidly identify transgenic animals. Before you submit your DNA for microinjection into fertilized mouse or rat eggs, the Core requires that you provide evidence that you have a PCR or Southern blot assay that detects your transgene when it is spiked into tail DNA at a one copy concentration. A second assay that will detect an endogenous mouse gene, such as beta-globin , or an endogenous rat gene, such as prolactin , is required in order to demonstrate that the DNA preparations are amenable to PCR. Animals are tested with both assays so that no transgenic founder is mistakenly discarded because the tail DNA is not suitable for PCR. You also need to have a Southern blot assay so that you can determine the copy number, integration site number, and transgene integrity in the transgenic founders prior to breeding.

Saunders, 2003.

microinjection DNA . Please note, if you want use large DNA fragments such as bacterial artificial chromosomes, that there is a specific protocol for the preparation of the BAC DNA for microinjection. Numerous publications show that BACs containing prokaryotic vector sequences are expressed at physiological levels in transgenic mice. Unlike plasmid based transgenes, removal of vector sequences in not required for BAC transgenes. Transgene constructs are then quantitated and microinjected into (C57BL/6 X SJL)F2 mouse eggs and surgically transferred to recipients. This is a fee for service provided by the Transgenic Core. The Transgenic Core prioritizes all requests for service on a "first-come, first-serve" basis. Your DNA will be added to the microinjection queue in the order that it is received. I you require a custom mouse strain we may be able to accommodate your needs. However, there is a surcharge for custom strains since transgenic production efficiency is lower in most custom strains.

DNA is extracted from the tail biopsies by simple salt out methods or you can use kits from various vendors on the market. For speed, some people use the "HotShot" method. Once the investigator has the DNA we expect you to test each DNA sample for both the transgene and an endogenous mouse gene or rat gene by PCR . Ideally, the you will identify which pups are transgenic before they are weaned at three weeks of age so that only transgenic pups are moved to your animal room. If the testing is not complete then we will transfer all of the pups to your animal room.

breeding . Transgenes typically insert in a head-to-tail concatemer. Thus, if you choose a restriction enzyme that cuts once in the transgene you will release DNA fragments the same size as the transgene from multicopy concatemers. The intensity of the hybridization signal will correspond to the copy number of the transgene in the insertion site (see copy standards ). Hybridization probes that bind to DNA fragments at the ends of the concatemer will be of unpredictable size since only one of the two restriction sites defining the DNA fragment will be in the transgene. If transgene arrays have integrated onto more than one chromosome the Southern blot will show multiple end fragments corresponding to the number of integration events. The frequency of this occurrence is around 10% of transgenic founders and is usually accompanied by the appearance of a very high copy number on the Southern.

9. Breeding and Analysis of Transgenic Rodents . The final stage in the process is to study animals carrying the transgene. Typically, the transgenic founder animals are bred to mice of defined genetic background such as C57BL/6. Transgenic rats are bred to Sprague-Dawley since that is their originating genetic background. Analysis of transgene expression and the consequences of expression is generally conducted in the offspring. The best strategy, if applicable is transgenic founder analysis. This eliminates the time and cost of breeding multiple offspring from each founder.