Recombinant Adenovirus in Neurobiology - Viral Vector Approaches in Neurobiology and Brain Diseases - page 14

2.4 Electroporator
and Electroporation
of E. coli Cells

Because of the low transformation effi ciency of BJ 5183 cells and
large size of AdEasy1 vectors, electroporation is highly recom-
mended for generating adenovirus recombinants. In general, elec-
troporation is carried out under the condition of 2.5 kV, 200 Ω , and

25 μ FD in an ice-cold 2 mm cuvette.

Electroporator: Bio-Rad Micropulser or other electroporators.

A stereotaxic apparatus for animals, such as rats and mice, is
required for injection of high-titer recombinant adenovirus
into selected brain regions.
Syringe pump for control the injection rate and volume. Syringe
pumps, such as a two-syringe push-pull pump or microsyringe
pump with four-channel microcontroller from Would Precision
Instruments (Sarasota, FL).

2.5 Materials for
Stereotaxic Injection
into the Brain

10 l Hamilton syringes .

Injector (33 gage, C315I, Plastics One Inc., Roanoke, VA), Guide
cannulas and PE50 tubing.
Slide-A-Lyzer mini dialysis unit (10,000 MWCO, No. 69570,
PIERCE).

μ l or 25 μ

3 Methods

3.1 Strategies
for the Design of
Recombinant
Adenovirus

In order to design a recombinant adenovirus suitable for your
research purposes, it is critical to choose the most appropriate
shuttle vector.
As listed in the material section, there are four shuttle vectors
in the AdEasy system. pAd-track vectors (with and without CMV
promoter) contain a GFP sequence that facilities visualizing viral
expression during generation of the virus and for various applica-
tions. Furthermore, GPF is a neuronal tracer that can move along
axons of infected neurons, which may be benefi cial for studies on
neurocircuitries. However, the inclusion of GFP sequence reduces
the capacity for insertion of a gene of interest. Thus, If an insert is
larger than 5.9 kb, pShuttle vectors (maximal insert size = 6.6 kb)
should be used. Additionally, the fl uorescence of GFP may interrupt
endpoint measurements such as immunohistochemistry and Ca 2+
release assays ( see Note 1 ).
Another consideration is whether to use vectors with CMV
promoter or not. pShuttle-CMV and pAd-track-CMV vectors
contain a CMV promoter that controls the expression of the gene
of interest. CMV is a human viral promoter with a high effi ciency
for viral expression. The CMV shuttle victors also contain SV40
poly A tail. These structures make construction of the adenovirus
much easier. However, the CMV promoter drives nonselective
expression of the gene of interest. The gene expresses in all of
the infected cells regardless whether it is endogenous expressed in