Transgenic Mouse General
Transgenic mouse generation has become relatively simple and low-costing now. Although it is still more expensive than tissue culture, mouse transgenics provides much more information and the data produced from mouse transgenetics are relative more applicable and reliable.
To make a transgenic mouse, most of the time, you will need a tissue-specific promoter, a functional 3\'UTR (which sometime is included in the promoter construct) and the open reading frame of your favorite gene. For almost all cell types, you can easily find well-defined promoters. However, the efficiency and specificity may not be as good as the endogenous gene. Bear that in mind when analyzing the phenotype of your transgenic mouse and interpreting your results. For an example, the widely-used Rat Insulin II promoter (Rip II) is not only active in pancreatic beta cells, but also (sometime unfortunately) in the brain. The Mouse Insulin I promoter (MIP) is more restricted to pancreatic beta cells. However, the specificity may vary based on the genomic integration site of the transgene. For an example, we found one line of the MIP transgenic mouse also expresses the transgene in intestine except beta cells.
The 3\'UTR is another aspect which you may pay extra attention to. Some 3\'UTR from commercial plasmids may function as expected in tissue culture, but not in vivo in mouse. For an example, we found the intron of SV40 3\'UTR from pBeta Gal Basic (Clontech) can not fully processed when used in transgenic mouse. The one 3\'UTR we have been using in multiple occasions is rabbit beta globin 3\'UTR.
After obaining the founder transgenic mouse (F1) from our transgenic facility, we usually perform a PCR reaction to identify positive lines. Then, we corss each individual line to next generation (F2) and after that we cross again (F3). Usually only after F3, we begin characterization since there may be a risk of losing the transgene before multiple generations. The first experiment we typically do is real time PCR or quantitative Western blotting to identify the high, medium and low expressors. After that, we usually take one animal from each line (high, medium and low) and harvest multiple tissues (more than 10) to determine the expression specificity. After all these initial characterization, we choose one or two lines for functional phenotyping.