David A. Zarling

Increased efficiency of transgenic livestock production

Production of transgenic livestock by pronuclear microinjection of DNA into fertilized zygotes suffers from the compounded inefficiencies of low embryo survival and low integration frequencies of the injected DNA into the genome. These inefficiencies are one of the major obstacles to the large-scale use of pronuclear microinjection techniques in livestock. We investigated exploiting the properties of recombinase proteins that allow them to bind DNA to generate transgenic animals via pronuclear microinjection. In theory, the use of recombinase proteins has the potential to generate transgenic animals with targeted changes, but in practice we found that the use of RecA recombinase-coated DNA increases the efficiency of transgenic livestock production. The use of RecA protein resulted in a significant increase in both embryo survival rates and transgene integration frequencies. Embryo survival rates were doubled in goats, and transgene integration was 11-fold higher in goats and three-fold higher in pigs when RecA protein-coated DNA was used compared with conventional DNA constructs without RecA protein coating. However, a large number of the transgenic founders generated with RecA protein-coated DNA were mosaic. The RecA protein coating of DNA is straightforward and can be applied to any species and any existing microinjection apparatus. These findings represent significant improvements on standard pronuclear microinjection methods by enabling the more efficient production of transgenic livestock.