Improved methods for tag and exchange recombineering

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BAC recombineering has revolutionized the generation of gene targeting vectors (TVs). We previously generated over 750 TVs using a combination of high throughput recombineering and Gateway technology. We now propose to develop a TV construction system that would leave the TV sequences seamless. Based upon our experience, there are two key technical problems that interfere with the efficient generation of TVs via tag and exchange recombineering using positive/negative markers. Conventional negative markers do not work effectively for exchange recombineering, as large numbers of false positive BAC clones result, which have undergone illegitimate recombination, deleting the marker without exchange. Second, replacer DNA used to exchange the tag is constructed by PCR, and prone to sequence error, even using high fidelity polymerases.