Preliminary testing of the original fly lines that we had hoped to use showed insufficient expression of the incorporated mitochondrial Green Fluorescent Protein (Mito-GFP) reporter. We have developed several new fly lines with both Mito-GFP and Atg8a mCherry reporters. This entailed first engineering a universal driver (Actin-Gal4) in a fly line expressing UAS-Mito-GFP. We then incorporated the ATG8a-mCherry into this line by recombination. We now have 7 lines expressing both markers that appear stable and viable. We are starting lifespan studies to document viability. These lines have been sent to NASA Ames Research Center (NASA-Ames) for additional testing.

In collaboration with Assistant Professor (Asst. Prof.) Caroline Kumsta at Sanford Burnham Prebys Medical Discovery Institute (SBP), we have generated a C. elegans line with Mito-GFP and Atg8-mCherry reporters. These are being tested at Ohio for compatibility with the C. elegans Maintenance Medium (CEMM) that will be used on the International Space Station (ISS).

We now, in collaboration with Research Asst. Prof. Georg Vogler, have a good method for frozen tissue preparation for single nuclear RNA sequencing technology that is becoming the new standard for gene expression technology.

In co-ordination with Co-Investigators (Co-Is) at NASA Ames Research Center (NASA - Ames), we are generating and testing the fly reagents needed for the experiments. These include a line of flies with a mitochondrial Green Fluorescent Protein (GFP) reporter and an ATG8-mCherry reporter that will allow us to monitor mitochondrial structure and autophagy respectively. We are also working to generate a C. elegans line with equivalent reporters. Toward this end we have brought on board a collaborator, Assistant Professor Caroline Kumsta, who will provide one of the reagents for the C. elegans experiment and will assist in combining that line with an available mitochondrial reporter line.

We are conducting preliminary tests to see if we will be able to adapt new state-of the art technology to the gene expression portion of the experiment. Toward this end we have brought on board a second collaborator, Research Assistant Professor Georg Vogler, to explore the possibility of using a new single cell RNA sequencing technology that is becoming a new standard for gene expression technology.