1. Volume 16, Issue 3, Pages 494-499 (March 2008)
Adeno-associated Virus of a Single-polarity DNA Genome Is Capable of Transduction In Vivo 
Xiaohuai Zhou, Xinghua Zeng, Zhenghong Fan, Chengwen Li, Thomas McCown, R Jude Samulski, Xiao Xiao 
Molecular Therapy 
Volume 16, Issue 3, Pages (March 2008)
DOI: /sj.mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Schematic representation of adeno-associated virus (AAV) terminal repeat deletions and production of polarity vectors. (a) Resolution and packaging of pDD series of plasmid. The plasmid is resolved at the Holiday junction to form a no-end DNA, which is subsequently converted into single-stranded genomes through the AAV replication process. Single-stranded genomes with intact D sequences at the 3′-end are efficiently packaged into viral particles; whereas genomes with 3′-D deletion are expected to be deficient in packaging. The plasmid sequences in the boxed area are detailed below. (b) Structures of plasmids with deletions in the D sequences. pDDwt-CMV-GFP, pDDΔ8-CMV-GFP, pDDΔ12-CMV-GFP, pDDΔ15-CMV-GFP, pDDΔ18-CMV-GFP, and pDDΔ29-CMV-GFP were constructed as described in the Methods. This series of plasmids were otherwise identical except that they contained 0, 8, 12, 15, 18, and 29 base pair (bp) deletion, respectively in a novel 165-bp AAV terminal repeat from one end of the D element. Plasmid pDDΔ18-CMV-GFP(−) was otherwise the same as pDDΔ18-CMV-GFP except that the deleted terminal repeats in the two plasmids were in reversed orientation. pDDΔ18-U1a-GFP and pDDΔ18-U1a-GFP(−) were derived from pDDΔ18-CMV-GFP and pDDΔ18-CMV-GFP(−), respectively by replacing the cytomegalovirus (CMV) promoter with the U1a promoter. GFP, green fluorescent protein; ITR, inverted terminal repeat.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Determination of the polarity of adeno-associated virus (AAV) vector genome. AAV vector DNA was extracted and dot-blotted to two identical sets of membrane as described in the Methods. The two sets of membrane were then hybridized each with one of the two complementary oligonucleotides derived from the cytomegalovirus (CMV) promoter, Probe CMV(+) or Probe CMV(−), which detect either the plus-strand or the minus-strand of the AAV DNA genomes. The amount of each polarity of the vector genome was determined by comparison with double-stranded, CMV-promoter plasmid standards loaded on each set of the membrane. GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Transduction of mouse muscle by single-polarity adeno-associated virus (AAV) vectors. The following five preparations of AAV vector at a dose of 1011 physical particles per animal were injected into the hindlegs of C57BL/6 mice: AAV-DDΔ18-CMV-GFP, AAV-DDΔ18-CMV-GFP(−), AAV-DDΔ18-CMV-GFP mixed with AAV-DDΔ18-CMV-GFP(−) in a 1:1 ratio, AAV-DDwt-CMV-GFP, and a control AAV-CMV-LacZ. Two weeks later, expression of the green fluorescent protein (GFP) transgene contained in the AAV vectors was imaged as described in the Methods. CMV, cytomegalovirus.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Transduction of mouse brain by single-polarity adeno-associated virus (AAV) vectors. The following three preparations of AAV vector at a dose of 1011 physical particles per animal were injected into the striatum of Sprague–Dawley rats: AAV-DDΔ18-CMV-GFP, AAV-DDΔ18-CMV-GFP(−), and AAV-DDΔ18-CMV-GFP mixed with AAV-DDΔ18-CMV-GFP(−) in a 1:1 ratio. Two weeks later, expression of the green fluorescent protein (GFP) transgene contained in the AAV vectors was examined as described in the Methods. CMV, cytomegalovirus.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Transduction of mouse liver by single-polarity adeno-associated virus (AAV) green fluorescent protein (GFP) vectors. (a) The following four preparations of AAV vector at a dose of 3 × 1011 vector genome (v.g.) particles per animal were injected into C57BL/6 mice through the portal vein: AAV-DDΔ18-U1a-GFP, AAV-DDΔ18-U1a-GFP(−), AAV-DDΔ18-U1a-GFP mixed with AAV-DDΔ18-U1a-GFP(−) in a 1:1 ratio, and a control AAV-CMV-LacZ. Six weeks later, expression of the GFP transgene from the AAV vectors was directly imaged as described in the Methods. (b) Lower vector doses (1 × 1011 v.g. particles) were identically administered and processed as in a, except that an immunofluorescent staining for GFP was also performed on consecutive sections to confirm the GFP expression.
Molecular Therapy  , DOI: ( /sj.mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions