Transgenic research

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Shepster
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Transgenic research

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. Areview 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.

Resources:
http://www.med.umich.edu/tamc/tgoutline.html
http://www.scienceclarified.com/Bi-Ca/Biology.html
http://www.pbs.org/wgbh/harvest/engineer/transgen.html