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Figure 2 | Genetic Vaccines and Therapy

Figure 2

From: The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

Figure 2

The expressed ribozyme RB15 in HepG2 cells. HepG2 cells (5 × 105 cells/well) were infected with rAAV-TTR-RB15 (1 × 1010 particles/well). (A). Total RNA was extracted from infected cells at days 3 (lane 2) and 7 (lane 3). Ribozyme RB15 RNA was detected using RT-PCR, followed by Southern blot analysis. Lane 1 is non-infected control RNA and lane 4 is H2O blank. For a negative control, the same experiment was performed also by PCR only. The DNA marker is 100-bp DNA ladder. The same experiment was carried out with the rAAV-TTR-RB15 mutant. The results are not shown here. (B). Total RNA (10 µg) was hybridized with 32P-UTP-labeled anti-apoB RNA and 32P-UTP-labeled anti-GAPDH RNA. The expression levels of apoB mRNA and GAPDH mRNA were determined by an RNase protection assay using an RPA III kit. After RNase digestion, the protected fragments (apoB RNA = 640 nt and GAPDH RNA = 316 nt) were generated and analyzed with 5% polyacrylamide gel electrophoresis. The concentrations of protected RNAs were determined by a Phosphoimager FX system (Bio-Rad). The levels of apoB RNA were normalized with GAPDH RNA and a representative experiment is shown here. The probes and protected fragments of apoB mRNA and GAPDH RNA are indicated.

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Genetic Vaccines and Therapy

ISSN: 1479-0556