Gene Therapy Strategies: Constructing an AAV Trojan Horse - Genomics: Essential Methods - page 287

(a) (b)
Figure 12.1 AAV-GFP transduction. The green fluorescent cells indicate the gfp expression ana-
lyzed by confocal microscopy ( × 250) (Leica Microsystems-TCS SP5). (a) Transduction efficiency
was 10% for MOI 100. (b) Transduction efficiency was 40% for MOI 1000. A vector pUF 11 (an
AAV-based plasmid vector) consisting GFP (535 bp) was transduced in human fetal fibroblasts
(IMR-90). IMR-90 cells were plated in six-well plates (5 × 10 4 cells/well) and were cultured in
minimum essential medium (MEM Engle) supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 0.1 m M non-essential amino acids and 1.0 m M sodium pyruvate. The cells were
cultured for 24 h at 37 ◦ C, 5% CO 2 . When cells were approximately 70-80% confluent, the cells
were washed in PBS and transduced with AAV-GFP vector. (See Plate 12.1.)

the selected cell marker transgene so that the transgene can be observed to be expressed in
one type of cell.

12.1.4 Production, purification and titration of recombinant
adeno-associated virus (rAAV)

The parvovirus AAV has a single-stranded genome of approximately 4.7 kb. rAAV in which
the two open reading frames of AAV, designated rep and cap, have been replaced by a gene
of interest has become an important tool for gene delivery [32].
The production method described in this chapter was developed by Muzyscka and
co-workers [33]. The protocol used is the rAAV plasmid (pTR-UF 3 ) [33] as an example to
describe the steps. The rAAV plasmid (pTR-UF 3 ) contains the GFP gene under control of
cytomegalovirus (CMV) promoter through the polio virus type 1 internal ribosomal entry
site (IRES) (Figure 12.2) and the helper plasmid pDG [34] which contains both the AAV
genes (rep and cap) for AAV propagation.

AMP R ITR CMV IRE GFP ITR

Figure 12.2 Plasmid (pTR-UF3) containing a CMV promoter with a GFP reporter gene. The
plasmid was developed at the University of Florida. ITR: AAV inverted terminal repeats; AMP R :
Ampicillin as antibiotic; other abbreviations in text.