Abstract
Here, we describe a method of systematic PCR screening with multiround sample pooling for the isolation of rare PCR-positive samples. As an example, we have applied this protocol to the recovery of gene-targeted clones in human somatic cells comprising only 0.02–0.17% of cells transduced with targeting vectors. Initially, cells infected with targeting vectors are seeded and grown in fourteen 96-well tissue culture plates. Samples are then collected from these plates and subjected to two rounds of pooling to yield twelve 'superpools' used for an initial PCR. After identifying PCR-positive samples, de-pooling is carried out with successive rounds of PCR screening, using samples of decreasing complexity. Single-cell cloning is subsequently performed to isolate gene-targeted clones. The entire protocol can be completed in 4–8 weeks depending on the proliferative capacity of the cell line.
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Acknowledgements
This work was supported by The Flight Attendant Medical Research Institute (FAMRI), NIH/NCI CA109274, The Elsa Pardee Foundation, the Summer Running Fund and the Avon Foundation. H.K. is a recipient of a Young Clinical Scientist Award from FAMRI and was also supported by the YASUDA Medical Research Foundation and the Kanzawa Medical Research Foundation. J.L. receives support from the American Society of Clinical Oncology's Young Investigator Award and is a recipient of a Young Clinical Scientist Award from FAMRI. J.P. Garay is a recipient of a Research Supplement to Promote Diversity in Health-Related Research, A.M.A. is supported by an NIH Institutional Training Grant T32CA67751 and J.P. Gustin is a recipient of a Department of Defense Breast Cancer Research Program Predoctoral Fellowship Award W81XWH-06-1-0325.
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Supplementary Video 1
Demonstration of Sample pooling round 1 (generation of 84 Pools) and round 2 (generation of 12 Superpools) procedures (MPG 19402 kb)
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Konishi, H., Lauring, J., Garay, J. et al. A PCR-based high-throughput screen with multiround sample pooling: application to somatic cell gene targeting. Nat Protoc 2, 2865–2874 (2007). https://doi.org/10.1038/nprot.2007.409
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DOI: https://doi.org/10.1038/nprot.2007.409
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