Biology Reference
In-Depth Information
Fig. 2
Analysis of the integrity and identity of the vector genome by multiple
restriction enzyme digestion. Informative restriction enzymes must be previously
chosen with a Sequence Analysis computer program
1. Digest 1
g of purifi ed plasmid DNA from the colonies selected
after homologous recombination with 0.3-0.5 U of an infor-
mative restriction enzyme, for 4 h.
2. Perform digestions with at least seven or eight different
enzymes and run in a 1 % agarose gel (
see
Fig.
2
).
3. If one restriction enzyme pattern does not correspond with the
expected pattern, repeat the digestion. If the observed pattern
still does not correspond with the expected pattern or if there
are more than one unexpected enzyme patterns, discard the
selected DNA.
4. Inoculate one positive recombinant in 200 mL of LB +
Ampicillin. Purify plasmid DNA with a commercial DNA
maxipreparation kit and store at −20 °C.
μ
3.2 Generation
and Amplifi cation
of Recombinant
Adenovirus
Recombinant adenovirus vectors are replication-defective and,
therefore, they must be produced and propagated in cell lines that
complement the defect. Even so, these vectors must be handled
following Biosafety Level 2 practices, just like the wild-type adeno-
viruses from which they are derived. The reason is that replication-
competent adenoviruses (RCA) can result from rare recombination
events between the recombinant vector genome and the adenoviral
sequences present in HEK293 cells during the amplifi cation pro-
cess. Appropriate information and guidance can be obtained from
each institution's Biosafety Committee and/or the Occupational
& Environmental Safety Offi ce.
