Beyond NEPTUNE (Neural plate targeting with in utero nano-injection): Targeting new organ systems to Reduce the numbers of mice used in science.

This proposal describes further adaptation of a method to replace traditional mouse genetics approaches, dramatically reducing the numbers of mice used in science, while offering superior results.

Thanks to two previous successful VR 3R proposals, we developed NEPTUNE (neural plate targeting with in utero nano-injection) to genetically manipulate up to 99% of cells in the developing brain, using ultrasound-guided in utero nanoinjection. The injections are similar to amniocentesis, performed on pregnant women, as the needle does not contact the embryo.

This method can dramatically reduce the numbers of mice used in science by generating only the required mice. For comparison: in traditional triple knockouts only 1 in 64 offspring is triple homozygous from heterozygous parents.

The suggested technology bypasses these problems, by specifically generating only the required mice.Here, we propose to further develop the technology to target the hematopoietic system, and the inner ear.

Our data show that we can target these systems, and we will now optimize efficiency for maximum transduction and clonal transduction.

To demonstrate the power of in utero injection to reduce the numbers of mice needed to resolve major questions in biology, we will perform lentiviral barcode lineage tracing, and CRISPR barcode lineage tracing experiments in each system.

This new technology thus holds great promise to reduce the numbers of mice used, while increasing the relative data output per mouse.