- Technical Note
- Open Access
An improved PCR strategy for fast screening of specific and random integrations in rAAV-mediated gene targeted cell clones
© Luo et al; licensee BioMed Central Ltd. 2011
Received: 20 April 2011
Accepted: 21 July 2011
Published: 21 July 2011
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. 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.
In 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.
This 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.
We would like to thank Trine Skov Petersen for skilled technical assistance. The project was supported by grants from the ''Pigs and Health" Platform of the Danish National Advanced Technology Foundation (Højteknologifonden) and from the Danish Agency for Science, Technology and Innovation (grant no. 274-05-0535).
- Luo Y, Li J, Liu Y, Lin L, Du Y, Li S, Yang H, Vajta G, Callesen H, Bolund L: High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer. Transgenic Res.Google Scholar
- Kohli M, Rago C, Lengauer C, Kinzler KW, Vogelstein B: Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res. 2004, 32: e3-10.1093/nar/gnh009.PubMedPubMed CentralView ArticleGoogle Scholar
- Rogers CS, Hao Y, Rokhlina T, Samuel M, Stoltz DA, Li Y, Petroff E, Vermeer DW, Kabel AC, Yan Z, et al: Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer. J Clin Invest. 2008, 118: 1571-1577. 10.1172/JCI34773.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.