Construct the Lenti-EGFP-β-catenin plasmid. Enzyme digestion of the plasmid.
Figure 1: Enzyme digestion of Lenti-EGFP-β-catenin plasmid. Sample 6-8 followed the predictive results
To verify whether Lenti-EGFP-β-catenin can work well in HEK293T cells or not, we do transient transfection, and the results show it works.
Package virus and infect cells to transfer EGFP-β-catenin into them.
Do sorting analysis, and find the infection failed. The reason may be the ratio of three kinds of packaging plasmids or other factors. Try the same ratio as well as experimental environment but more carefully again.
The results of the second trial are not good. So we try other ratios of three packaging plasmids and do other two experiments. Finally find the ratio of PMD2.G to PSPAx2 to vector equals to 1:1:1.5 is appropriate.
Use the appropriate ratio to infect cells with mCherry.
qPCR for all of those infected cells to see whether the sequence of interest (mCherry) is integrated into their genome or not, and the result is good.
FACS sorting and pick out cells infected successfully, and also divided into two types, S1R-low and S1R-high, depend on their signal strength without Wnt.
Add 3μM Chir99021( a drug that inhibits GSK3β and has similar function with Wnt ligand) to S1R-low and S1R-high, and do flow analysis after different incubating time (0h,3h,6h,9h,12h, and 24h) to find out the ideal time length of drug.
Figure 2:The response strength of S1R-Low and S1R-Hight cells in series of time.
Do flow sorting after adding drug and divide cells into S2R-low and S2R-high.We named cells with low signal after drug P6, and high signal P4.
Coculture P6( low signal after drug) with Lwnt3a cell, it shows low signal response after 24 hours, which means both it works well.
Package Lenti-Cas9-Blast virus and infect Lwnt3a cells. Change the supernatant after 24h, and then add Blasticidin (Blast) after another 24h to kill cells without Cas9 expression, use no treated Lwnt3a cells as negative control.
Package Lenti-Cas9-mCherry virus, infect Lwnt3a cells.
Flow sorting, to pick out Lwnt3a cells transferred Cas9-mCherry successfully. Because this method is much better than use Cas9-Blast and Blasticidin screening, we choose Lwnt3a-Cas9-mCherry as our donor cell line.
We repeat the experiments of building Lwnt3a-Cas9-mCherry cell line because the former cells grew badly and dead mostly.
Repeat the flow sorting and get new Lwnt3a-Cas9-mCherry cells.
Because at the very end of our experiment we are going to put cells into double emulsion, we need cells that can survive in suspension system. We do suspension test with HEK 293E cells, with 130 rpm rotation speed, 10ml medium and 2.82x10^5 cells in total(1/25).
Cut Lenti-Guide-puro as backbone plasmid with enzyme and stay overnight.
Run the product of enzyme digestion and harvest the ideal part, which’s length is 8298pb.
Figure 3:Gel data after enzyme digestion of Lenti-Guide-Puro plasmid
Count 293E cells in suspension test: 7.51x10^5/ml in total, and death rate is 11.72 %( 1/30). 5.57x10^5/ml and alive 1.37x10^5/ml (2/2)
Ligate Guide-puro backbone plasmid with single guide RNA of Porcupine1, Porcupin2, Wls1, Wls2, Non-target1, Non-target2, and wnt3a1, and in order to avoid low efficiency, for some genes, we design two different guide RNA as well as control.
We draw growing curve to see how S1R grows in suspension state: 24h: 10.8x10^5 cells, 48h: 1.615x10^6 cells.
Growing curve: 60h: 1.38x10^6/ml, 72h: 1.61x10^6/ml, 96h: 1.31x10^6/ml (most cells only grow in one area of the well, so the number may inaccurate.)
Figure 4:Growth curve of 293T suspension culture in a flask
Because we need cells that can work in suspension environment, we do suspension training to 293T cells. The total volume of medium is 25ml, and density of cells is 3x10^5/ml, the medium is mixed by DMEM (makes up 75%) and Union 293 (makes up 25%).
TCF viruses packaging, and harvest after 48h.
Do Mycoplasma detection to Lwnt3a-Cas9-mCherry cells, the result is positive so we add drugs to solve the issue.
Suspension training: change the supernatant from 75% DMEM+ 25% Uninon293 to 40% DMEM+ 60% Union293.
Because the results of former Lwnt3a-Cas9-mCherry cells are not good, we repeat relative experiments and finally get new Lwnt3a-Cas9-mCherry cells. This time, we culture these sorted cells with antibiotics P/S (1:100) and Plasmocin (Mycoplasma elimination reagent) (1:500).
Count 293E cells in suspension test: 30000 cells with 85% death rate (4/3), 35200/ml with death rate 95% (4/4).
There is fungal contamination in 293T cells in suspension training, so we sterilize and discard it.
Harvest the supernatant of Lwnt3a with different time length: 12h, 24h, and 72h. Do Western blot by using former samples as well as product from Lwnt3a cells which are suspension for 24h, and Wnt standard samples with concentration of 50, 100, 200, 400ng/ml.
Figure 5:Western blot data
Package viruses with Non-target, Porcupine, Wls, and Wnt as target genes in 293T cells. Change the supernatant after 24h and harvest viruses after another 24h.
293E cells in suspension test: 6.08x10^6/ml (4/17), 7.36x10^6/ml (4/18), 1.952x10^7/ml (4/19), 1.36x10^6/ml (4/20).
Infect Lwnt3a-Cas9-mCherry cells with gRNA-puro viruses. Add Puromycin to screen out cells infected successfully.
293E cells in suspension test: 2.38x10^6/ml (4/24), most of cells grows individually and separately.
293E cells in suspension test: 2.4x10^6/ml (5/4), 3.28x10^6/ml (5/5).
293E cells in suspension test: 6.56x10^6/ml (5/6), 6.16x10^6/ml (5/7), 8.88x10^6/ml (5/8), 6.48x10^6/ml (5/9) and most cells grow in groups, 9.2x10^6/ml (5/10) and most cells grow in groups, 1.072x10^7/ml (5/11).
Figure 6:Growth curve of 293E suspension cell line
Western blotting with supernatant of 72h adherent Lwnt3a cells, suspended Lwnt3a cells, supernatant of 6h/12h/72h Lwnt3a-mCherry cells, and 640/320/160/80/40/20μg/mL Wnt standard samples.
Figure 7:Western blot data
Do flow analysis to 293E, 293E-TCF, and P4 cells with chir or Wnt, to see the differences.
Add different concentration of Wnt and R-spondin(ligand that enhance WNT pathway activity) to 293E, 293E-TCF and P4, and do flow analysis after 24h.
Use different concentration of Wnt and R-spondin as well as adding R-spondin early or not to 293E, 293E-TCF and P4. Do flow analysis after 24h.
Add 3μM chir to 293E-TCF P2 and P8, and do flow analysis after 24h, in order to see if we have made mistake by storing 293E as 293E-TCF.
Grow other cells while discard R-spondin cells due to its bad state.
Do Mycoplasma detection to 293E, 293E-TCF, and P4. The results are positive so we add drugs to those cells’ culture medium.
Do experiments to see the best condition for cells in the double emulsion machine.
Figure 8:PI staining of cell culture mimic droplet environments. (Default conditions: 4°C, DMEM medium, 1.5mL tube, close lid and flow cytometry)