The use of recombinase proteins to generate transgenic large animals.

The endogenous properties of recombinase proteins allow them to associate with and bind DNA to catalyze homologous recombination. These endogenous properties of cellular recombination enzymes may be useful to the field of transgenesis. The production of transgenic animals, in particular livestock, is an inefficient process by both conventional pronuclear microinjection techniques and nuclear transfer. Furthermore, the use of pronuclear microinjection is currently limited to the random addition of genes and does not allow for the replacement of an endogenous gene with a more desired one. The functions of cellular recombination enzymes have been exploited to develop a technique that is compatible with pronuclear microinjection and may make the process of generating transgenic livestock more efficient while also enabling the targeting of homologous chromosomal genes. In our hands, transgenic animals generated by the pronuclear microinjection of various recombinase protein-coated DNA fragments led to a higher than expected birth rate as well as transgene integration frequency. Most founder animals generated were likely mosaic, indicating that integration occurred after cell division. The presence of multiple related genes makes detection of any recombination event difficult. Overall, this technique is a straightforward, rapid, and efficient procedure that can be applied to any segment of DNA and any microinjection apparatus, and is less labor intensive than nuclear transfer.