Abstract
BackgroundGene targeting by homologous recombination using recombinant adeno-associated virus (rAAV) is becoming a useful tool for basic research and therapeutic applications due to the remarkably high targeting frequency of rAAV virus vectors. However, the screening for the pure gene-targeted and random-integration-free primary cell clones is difficult since the cells have a limited proliferation capacity and often cannot be grown to produce sufficient DNA for non-PCR based analysis. This hampers the applications of this technology.FindingsIn this study, we have developed an improved PCR screening method, which can be used for fast screening of clones with unwanted random integration (RI) of the rAAV genome. This improved screening method includes four PCRs: a PCR for the selection gene (e.g. Neo-PCR), a PCR for targeted gene knockout (e.g. BRCA1-KO-PCR), and two generalized PCRs for random integration of the rAAV genome (5'-AAV-RI-PCR, and 3'-AAV-RI-PCR). We have shown that this screening method greatly facilitates the procedure of screening for BRCA1 (BReast CAncer susceptibility gene 1) targeted cell clones, eliminating cell clones with both BRCA1 knockout and random integration of the rAAV genome.ConclusionsThis screening method has facilitated the screening of correct gene-targeted cells. As the AAV-RI-PCRs are generalized PCRs, this method can also be applied for screening of rAAV-mediated targeting of other genes.
Highlights
Gene targeting by homologous recombination using recombinant adeno-associated virus is becoming a useful tool for basic research and therapeutic applications due to the remarkably high targeting frequency of rAAV virus vectors
As the AAV-RIPCRs are generalized PCRs, this method can be applied for screening of rAAV-mediated targeting of other genes
Primary fibroblasts are commonly used as target cells for gene targeting prior to cloning by somatic cell nuclear transfer
Summary
Gene targeting by homologous recombination using recombinant adeno-associated virus (rAAV) is becoming a useful tool for basic research and therapeutic applications due to the remarkably high targeting frequency of rAAV virus vectors. Conclusions: This screening method has facilitated the screening of correct gene-targeted cells. As the AAV-RIPCRs are generalized PCRs, this method can be applied for screening of rAAV-mediated targeting of other genes.
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