Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 13

BJ5183 Cells Containing AdEasy-1 Vector (AdEasier or BJ5183-Ad-1
Cells)

BJ5183 cells have high capability for homologous recombination and thus
are used for recombination of the shuttle vector and AdEasy-1 vector.
However, these cells have a relatively low transformation effi ciency.
The success rate is low when both shuttle vector and AdEasy-1 vector
have to be transfected together and then are recombinated. A more
recent approach is to fi rst transfect AdEasy-1 into BJ5183 cells and then
to transfect the shuttle vector into BJ5183 containing AdEasy-1. This
approach for obtaining recombinant adenovirus is much more effi cient.
BJ5183 cells containing the AdEasy-1 vector are called AdEasier or
BJ5183-Ad-1 cells (Stratagene).

HEK 293 or 291 Cells

HEK 293 and 291 cells contain an E1 domain of adenovirus that is
required for viral propagation. Thus, HEK 293 or 291 cells are used to
generate recombinant adenovirus.

2.3 siRNA
Recombinant
Adenovirus System

To generate recombinant adenovirus containing siRNA, two vectors
were developed, one for identifi cation of siRNA (pSOS-HUS) and
one, a shuttle vector, for recombination of siRNA into adenovirus
(pSES-HUS) [ 15 ].
pSOS-HUS vector is designed for identifying siRNAs for a gene
of interest. The vector contains an siRNA site that is controlled by
U6 and H1 promoters, and a target gene site (MCS) that is at 3

′

of an eGFP sequence and linked to the eGFP with an IRES pro-
moter, resulting in the expression of eGFP and the gene of interest,
whereby they are transcribed together but are translated separately.
When an siRNA binds to mRNA of the gene of interest and con-
sequently results in degradation of the mRNA, the eGFP mRNA
is also degraded. Therefore, the expression of eGFP is negatively
correlated to the effect of the siRNA in the SOS-HUS. The design
to include both siRNA and the sequence of the gene of interest in
one vector results in transfection of the siRNA and gene of interest
simultaneously.
pSES-HUS is a shuttle vector for siRNA. The siRNA site is
controlled by U6 and H1 promoters. A double-stranded DNA oli-
gos with the siRNA sequence is inserted between Sfi I sites. Using
a DNA oligo cassette signifi cantly reduces the cost relative to the
cost for siRNA. The pSES-HUS contains an RFP sequence that
assists in visualization of the expression of the recombinant
adenovirus.