Our project is based on Wnt signaling pathway and microfluidic double emulsion system. We focus on key genes in Wnt secretion and develop a high-throughput way for studying it. In detail, we constructed genetically engineered donor and reporter cell lines. Donor cells are Wnt-secreting cells with CRISPR-Cas9 gene knockout. Reporter cells can detect Wnt signal and emit green fluorescence. Two type of cell lines are cocultured in 30um microdroplets by double emulsion system. Few thousand droplets can be generated in 20 minutes, which is a powerful method. We can study signal transduction according to the detecting fluorescence to select the potential key genes in Wnt signaling. We demonstrated experimental design for proof-of-principle.
Step One: Cell line construction
We constructed two cell lines: Wnt-secreting cell and Wnt-responding cell.
Wnt secreting cell came from mouse L-wnt3a cell (L cell), it was modified and stably secreted Wnt3a. It was then transfected a Cas9-mCherry construct and a single guide RNA insert through Lentiviral infection. Applying CRISPR-Cas9 screening system, we can construct multiple secreting cell lines with one specific gRNA transfected. In our experiments, we only knockout 3 genes for proof-of-principle.
Wnt responding cells come from human HEK 293R, its genome was integrated with a TCF-GFP reporter system. TCF is a promoter which only bind to TCF7 transcription factor as a specific downstream effect of Wnt signaling.
Step Two: Encapsulation of cells by microfluidic systems
One Wnt-secreting cell and one Wnt-responding cell are encapsulated in a microdroplet by our microfluidic systems. We develop two parallel strategies for encapsulation: double emulsion droplets and micro-well plates methods.
Double emulsion has two steps of emulsion. The first emulsion is to encapsulate two single cells in a water-in-oil microdroplet. The second emulsion is to re-encapsulate the droplet into a water-in-oil-in-water (wow) droplet which is beneficial in Flow cytometer selection. The system can produce few million droplets in a size of 30 microns within 20 minutes. The encapsulation rate is quite good.
Step Three: Cell coculture in micro-environment
Two single cells are cocultured for few hours in a micro-environment.
When the Wnt signal is ON, reporter cell line can detect the signal and express GFP fluorescence. While no fluorescence is performed if the Wnt signal if OFF. The Wnt secretion level determined by the knockout effect of specific gene. We will screen the key genes in Wnt secretion through this methods.
To study the Wnt secreting pathway in Wnt signaling, multiple Wnt-secreting cells will be knockout with multiple genes and cultured with Wnt-responding cell in few thousand of micro-environments. It is then a series of parallel experiments done at a time for studying the genetic effects in Wnt secretion pathway.
Step Four: Target selection by FACS
After coculture, we can select the droplets according to consequential GFP fluorescence. Microdroplets produced by double emulsion system can be selected through Fluorescence-activated Cell Sorter(FACS) while micro-well plate can be picked by Microprobe platform based on the fluorescence signal strength.
Step Five: DNA sequencing for key gene screening
After selection, gene information is store in gRNA construct in the genome of secreting cells. It is possible to enrich the gRNA sequence through PCR. The PCR product can be sequenced with second generation sequencing. Key genes in Wnt secretion will be possibly found.