Traditional transgenes are defined here as transgenes cloned into bacterial vectors, such as pBluescript and pUC. These transgene constructions, not including the vector, are usually less than 10 kb. These constructions are usually coding regions fused to promoter elements. If you are attempting to express cDNA sequences, it would be helpful to include an intron and a good polyadenylation signal. In the design of these transgenes, the transgene insert must be isolated free from the bacterial expression vector. The purity of the transgene fragment to be injected is critical for efficient generation of transgenic mice. In order to ensure that the transgene fragment is "Microinjection Quality", the GEM Core will isolate the transgene unless the investigator has a specific reason to isolate the fragment themselves. The investigator is asked to follow the protocol below:
1. Digest 100 ug of plasmid with the appropriate restriction enzyme(s) in a 300 ul reaction volume.
2. Run an aliquot of the digest on an agarose gel and photograph the results.
3. Indicate on the photograph the band to be isolated along with the size of the fragment.
4. Attach photograph and Eppendorf tube containing the rest of the digest to the MEMs form and deliver it to N630. Note: The MEMS form must include the request number.
The transgene should be cut clean and free from vector. The transgene must be easily separated from the vector. For example: If the Vector is 2.9 kb and the transgene is 3.0 kb, it can not be easily separated on a normal gel. The vector must be cut with additional enzymes to allow at least a 1 kb molecular weight difference between the fragments. If you can't digest the vector free from the transgene, you may have to redesign the transgene construction. "Successful transgene experiments begin with the design of the transgene construction." If you have any questions regarding this aspect of transgene preparation contact Memsmail@bcm.edu.