Traditional transgenes are defined here as transgenes cloned into bacterial
vectors such as Bluescript 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 provide
the GEM Core with:
1.
100 ug of restriction enzyme digested plasmid in a 300 ul reaction volume
2.
Run an aliquot of the digest on an agarose gel and include that photograph
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 M725.
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 trangene construction."
If you have any questions regarding this aspect of transgene preparation contact
Dr. DeMayo.
