Question

What is the split gal4 system? Why is it needed to label some cell types? Describe an experiment that combines split gal4 with scRNA-seq to precisely define cell identities in the drosophila brain. Describe the protocol of the experiment in detail

Answer 1

The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly.The Gal 4 system allows separation of the problems of defining which cells express a gene or protein and what the experimenter wants to do with this knowledge.Gal4 expression can be made even more specific by means of "intersectional strategies". These can combine two different GAL4 lines—say, A and B—in a way that GAL4 is only expressed in the cells that are in line A but not line B, or those that are in both lines A and B. When combined with intrinsically sparse GAL4 lines, this offers very specific selection, often limited to a single cell type.To express GAL4 in only the cells contained in both A and B, a technique called "split-GAL4" can be used. Line A is made to express half of the GAL4 protein, which is inactive by itself. Similarly, line B is made to express the other half of GAL4, also inactive by itself. Only the cells that are in both lines make both halves, which self-assemble by leucine zipper into GAL4 and activate the reporter gene.

The Drosophila split-GAL4 system allows for restricted expression of regulatory targets only in cells where the two components of the split-GAL4 activator are co-expressed. the GAL4 DNA-binding domain fused to the Zip- protein-pairing domain can be expressed in one pattern, a transcriptional activation domain fused to the Zip+ protein-pairing domain can be expressed in another pattern and the UAS reporter construct will be expressed in the intersection of the two patterns. In this method, the coding region of the GAL4 gene is split and one enhancer is used to drive the expression of the GAL4 activating domain (AD) and a second enhancer is used to drive the expression of the DNA-binding domain (DBD).

EXPERIMENT: scRNA-seq are applied using droplet microfluidics (10x Chromium) on dissociated adult brains from animals precisely aged to eight different time points. To take genetic diversity between domesticated D. melanogaster strains into account, brains are dessected from two different lab strains. Using stringent filtering, 56,902 (57K) high-quality cells are retained from 26 runs. A larger dataset of 157K cells can be collected based on lenient cell filtering, combined with cells from three additional DGRP-551 runs that were performed with an alternative dissociation protocol . A median sequencing depth of 53,553 sequence reads per cell can be obtained, with median sequence saturation rate of 81.5%. The total number of genes detected across all cells are 12,436 protein-coding genes and 2,164 non-coding RNAs. The median number of genes per cell ranges between 1,308 (0 days) and 810 (50 days). The 57K filtered cells are clustered using Seurat, leading to 87–151 cell clusters, depending on the clustering resolution.The 57K dataset with 87 initial clusters can be used. These cells are subsequently visualized by generating t-distributed stochastic neighbor embedding (t-SNE) plots.