1. Volume 16, Issue 5, Pages 924-930 (May 2008)
Intron Splicing–mediated Expression of AAV Rep and Cap Genes and Production of AAV Vectors in Insect Cells 
Haifeng Chen 
Molecular Therapy 
Volume 16, Issue 5, Pages (May 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Schematic depiction of the genetic and transcriptional maps of adeno-associated virus (AAV) type 2 and recombinant baculoviruses Bac-inRep and Bac-inCap. (a) Illustration of the genetic and transcriptional map of adeno-associated virus (AAV) type 2 (GenBank accession no. AF043303). (b) Illustration of the artificial intron containing the polyhedrin (polh) promoter. (c) Illustration of the genetic and transcriptional map of a representative recombinant baculovirus (Bac-inRep) expressing both Rep78 and Rep52 of AAV type 2 within a single expression cassette. Mature Rep78 messenger RNA (mRNA) is formed when the artificial intron is removed through splicing. Rep52 mRNA is transcribed from the promoter located inside the artificial intron. The numbers on the genetic map correspond to the nucleotide number of AAV type 2 genome. (d) Illustration of the genetic and transcriptional map of a representative recombinant baculovirus (Bac-inCap) expressing VP1, VP2, and VP3 of AAV type 2 within a single expression cassette. Mature VP1 mRNA is formed when the artificial intron is removed through splicing. mRNA encoding VP2 and VP3 is transcribed from the promoter located inside the artificial intron. The numbers on the genetic map correspond to the nucleotide number of AAV type 2 genome.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Western blot analyses of adeno-associated virus (AAV) Rep and Cap protein expression in Sf9 cells. (a) AAV type 2 Rep78 and Rep52 expressions in Sf9 cells transfected with bacmid (lane 1) or infected with recombinant baculovirus (lane 2) carrying the artificial intron-containing Rep coding sequence; AAV type 2 VP1, VP2, and VP3 expression in Sf9 cells transfected with bacmid (lane 3) or infected with recombinant baculovirus (lane 4) carrying the artificial intron-containing Cap coding sequence. Each lane was loaded with lysate equivalent to 1 × 105 (lanes 1 and 2) or 5 × 104 (lanes 3 and 4) Sf9 cells. (b) Cap protein expressions of AAV types 1, 6, and 8 in Sf9 cells. Lanes 1 and 4, AAV type 2 VP proteins from Sf9 cells as control; lanes 2 and 3, AAV type 1 VP protein expression in Sf9 cells from two individual clones of recombinant baculovirus carrying the artificial intron-containing Cap coding sequence; lane 5, AAV type 6 VP protein expression in Sf9 cells infected with recombinant baculovirus carrying the artificial intron-containing Cap coding sequence; lane 6, AAV type 8 VP protein expression in Sf9 cells infected with recombinant baculovirus carrying the artificial intron-containing Cap coding sequence. Each lane was loaded with lysate equivalent to 5 × 104 Sf9 cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Western blot analyses of adeno-associated virus (AAV) types 2, 6, and 8 VP proteins in Sf9 cell lysates and purified AAV vectors. (a) VP proteins of AAV type 2 in Sf9 cell lysates and purified vectors. Lane 1, lysate of Sf9 cells triple infected by Bac-Rep, Bac-Cap, and Bac-GFP; lane 2, lysate of Sf9 cells triple infected by Bac-inRep, Bac-inCap, and Bac-GFP; lane 3, lysate of Sf9 cells triple infected by Bac-Rep, Bac-Cap, and Bac-RFP; lane 4, lysate of Sf9 cells triple infected by Bac-inRep, Bac-inCap, and Bac-RFP; lanes 5–8, purified vectors from Sf9 cell lysates corresponding to lanes 1–4; and lane 9, purified AAV type 2 vectors from 293 cells for comparison. Each lane was loaded with lysate equivalent to 5 × 104 Sf9 cells or purified vectors equivalent to 1 × 1010 vector genome copies (vg). (b) VP proteins of AAV types 6 and 8 in Sf9 cell lysates and purified vectors. Lane 1, lysate of Sf9 cells triple infected by Bac-Rep, Bac-Cap6, and Bac-GFP; lane 2, lysate of Sf9 cells triple infected by Bac-inRep, Bac-inCap6, and Bac-GFP; lane 3, lysate of Sf9 cells triple infected by Bac-Rep, Bac-Cap8, and Bac-GFP; lane 4, lysate of Sf9 cells triple infected by Bac-inRep, Bac-inCap8, and Bac-GFP; lanes 5–8, purified vectors from Sf9 cell lysates corresponding to lanes 1–4. Each lane was loaded with lysate equivalent to 5 × 104 Sf9 cells or purified vectors equivalent to 2 × 1010 vg. RFP, red fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Infectivity of AAV2-GFP and AAV6-GFP vectors produced by different methods. (a) 293 AAV2-GFP, AAV type 2 vector purified from 293 cells with triple-plasmid transfection method; Sf9 AAV2-GFP (ACGVP1), AAV type 2 vector purified from Sf9 cells by triple-infection method with recombinant baculoviruses carrying the intron-less Rep and VP1 start codon–mutated (AUG to ACG) Cap coding sequences, respectively; Sf9 AAV2-GFP (AUGVP1), AAV type 2 vector purified from Sf9 cells by triple-infection method with recombinant baculoviruses carrying the artificial intron-containing Rep and authentic AUG start codon–containing Cap coding sequences, respectively. (b) Sf9 AAV6-GFP (ACGVP1), AAV type 6 vector purified from Sf9 cells by triple-infection method with recombinant baculoviruses respectively carrying the intron-less Rep and VP1 start codon–mutated (AUG to ACG) Cap coding sequences; Sf9 AAV6-GFP (AUGVP1), AAV type 6 vector purified from Sf9 cells by triple-infection method with recombinant baculoviruses carrying the artificial intron-containing Rep and authentic AUG start codon–containing Cap coding sequences, respectively. AAV, adeno-associated virus; GFP, green fluorescent protein.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Western blot analyses of stability of recombinant baculoviruses. (a) Passage 5 lysates from 12 plaque-purified recombinant baculovirus clones carrying the artificial intron-containing Rep coding sequence. (b) Lysates from five different passages of one single recombinant baculovirus clone carrying the artificial intron-containing adeno-associated virus (AAV) type 2 Rep coding sequence. Lanes 1–3, passages 1–3 lysates from Sf9 cells infected for 2 days; lanes 4 and 5, passages 4 and 5 lysates from Sf9 cells infected for 3 days. (c) Passage 5 lysates from six plaque-purified recombinant baculovirus clones carrying the AUG start codon and artificial intron-containing AAV type 2 Cap coding sequence. (d) Lysates from five different passages of one single recombinant baculovirus clone carrying the AUG start codon and artificial intron-containing AAV type 2 Cap coding sequence. Lanes 1–3, passages 1–3 lysates from Sf9 cells infected for 2 days; lanes 4 and 5, passages 4 and 5 lysates from Sf9 cells infected for 3 days. Each lane was loaded with lysate equivalent to 1 × 105 Sf9 cells for the Rep and 5 × 104 Sf9 cells for the Cap analyses.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions