• Introduction
  • Experimental Timeline
  • Plan the Experiment
  • Clone the Transgene
  • Transgene Screening
  • Transgene Expression
  • DNA Microinjection
  • Identify Transgenic Founders
  • Southern Analysis
  • Transgenic Analysis
  • Pertinent Publications
  • Review Articles and Books
  • Top Home

    1. Introduction. This is a brief outline of the steps necessary to obtain transgenic mice or transgenic rats. Simply put, the investigator constructs a transgene with a promoter and a structural gene (for example a reporter gene such as lacZ or a transcription factor). DNA is prepared and microinjected into fertilized mouse eggs. Potentially transgenic mice or rats are born. Transgenic founders are identified, usually by a simple PCR assay,  and bred to produce offspring for analysis. Core personnel are available for consultation on all aspects of transgenic research. The transgenic founders are then bred to establish mouse lines and the offspring of each founder are tested for transgene expression. Contact Thom Saunders with any questions or Meet the Staff.

    2. Plan the experiment. What is the purpose of your experiment? Do you want to define tissue specific regulatory sequences? Do you want to overexpress a protein in a specific cell lineage? You will need to obtain or clone the desired promoter and structural gene. You will need to establish a gene expression assay. If you expect an observable phenotype in the transgenic mice you should establish methods to measure and quantitate the phenotype. Expression of some genes will be deleterious or incompatible with proper growth and development of the embryo. Special arrangements should be made if you expect embryonic lethality from transgene expression. The expression of a transgene requires that the appropriate transcriptional control elements be included in the DNA construct. A literature search may identify these elements. Preliminary studies in cell cultures are recommended to verify the integrity of the construct and the function of the promoter. However, it is not always possible to predict in advance whether the transgene will have the capability of being expressed in vivo. A 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.

    3. Clone and the Verify the Integrity of the Transgene. In transgene design several things should be considered during cloning. For exemple, prokaryotic vector sequences interfere with the expression of some transgenes, thus unique restriction sites at the 5' and 3' ends of the construct should be available for vector removal. The transgene should contain unique markers so that its presence can be easily detected in mouse tissue DNA samples and so that its expression can be assayed and distinguished from endogenous gene expression. Sequencing of junction fragments should be carried out in order to confirm that the transgene has a functional promoter, initiation codon, and polyadenylation signal. There are several reports that the inclusion of introns will increase transgene expression (see Review Articles). Under the best circumstances, the transgene is tested for expression in a cell  culture system before transgenic mice are made.

    4. Establish a Screening Method. We suggest that you establish a PCR assay to rapidly identify transgenic animals. Before you submit your DNA for microinjection into fertilized mouse or rat eggs, the Transgenic 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 free of PCR inhibitors. Animals are tested with both assays so that no transgenic founder is mistakenly discarded because the tail DNA is contaminated with PCR inhibitors. If your transgene is a bacterial artificial chromosome, it recommended that you use mutliple markers spread along the BAC to make sure an intact copy of the BAC is present in your transgenic founders. 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.

    5. Establish an Expression Assay. It's important to show that transgene is expressed. RNA expression can be detected by in situ hybridization or RNAse protection assay with RNA probes. Alternatively, an RT-PCR approach can be used. The protein that is produced must be different from proteins normally expressed in the mouse. There are several ways to achieve this. You may use a reporter gene such as a fluorescent protein that you can visualize directly. You may use a reporter gene that provides for enzymatic amplification of the signal such as beta-galactosidase or human placental alkaline phosphatase. You may use a non-mouse protein such as a human protein or an epitope tagged (i.e. HA, FLAG or myc) protein that can be identified with an antibody that doesn't bind to mouse proteins. For further discussion of reporters in transgenic mice refer to Saunders, 2003.

    6. DNA Purification and Microinjection. We will purify the DNA for you. Simply perform a restriction enzyme digest on your cloning vector to liberate 50 ug of the transgene insert from the cloning vector. Run out a few hundred nanograms of DNA on a minigel to determine whehter the digest went to completion and that the bands are the correct size. Bring the remainder of the digest (in a final volume of 100 to 150 microliters) to the Core lab and we will purify the DNA for microinjection from the digest.  We use the Nucleospin Extract Kit for purification of 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 (Van Keuren et al., 2009). 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 provided on a fee for service basis by the Transgenic Facility.. 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. If you require a different  mouse strain (e.g. C57BL/6J or FVB/NTac)  we may be able to accommodate your needs. However, there is a surcharge for custom strains since transgenic efficiency is lower in most custom strains.

    7. Screen Potential Founders. Tail biopsies from potentially transgenic mice and rats will be obtained 5 weeks after injecting eggs (3 weeks gestation time and 2 weeks of post-natal growth). You will need to extract DNA from the tail tips for use as a PCR template or for Southern blot analysis. The Transgenic Core expects you to test each DNA sample for both the transgene and an endogenous mouse gene or rat gene by PCR. Ideally, 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.

    8. Southern Analysis. Between the time that the transgenic pups are identified and they are 6 weeks old Southern blot analysis should be done to determine how many copies of the transgene integrated, how many chromosomal sites the transgene inserted into, to verify transgenic status and to determine if the transgene is intact. With this information, transgenic founders with a good chance of transmission (at least 5-10 copies) of an intact transgene in a single insertion site can be selected for intensive 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/6J. Transgenic rats are bred to Sprague-Dawley (Crl:CD (SD)) or other orignating 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.


    Top Home

    Transgenic Outline | Gene Targeting Outline | Laboratory Protocols |List of Services | Service Description
    Genotyping Mice |Mouse Breeding Suggestions |Links|Home


    comments to: tsaunder@umich.edu