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InbarC - 31 May 2011
6 month plan (November 2010)
Molecular
- Prepare constructs containing selection markers, fluorescent markers, and Wnt promoters/signals/targets/inhibitors or differentiation markers
- Insert constructs into ES cells.
Statues: have ready plasmids for differentiation, Beta-Catenin, DKK1, TCF. Have not yet prepared DOX induced Wnt signals (requires extensive molecular planning). Need to lift Wnt promoter sequences (not avaliable on market).
Roadblocks: need clean cells to transfect with the constructs.
Need to plan Wnt promoters to lift.
Cellular
- Select and store cell lines containing these markers
Statues: Nothing. Have stored R1 ES transfected with brachyury, Flk1 and alpha-MHC. But no succesful selection. So the cells are mixed.
Roadblocks: Need clean R1 cells. And clean Kh2 cells. Once I have them I can move forward.
EB differentiation
- Define Media+ chamber/plate (+plating protocol) leading to the development of CMs within relatively small (200-300um diameter) EBs. achieve reproducible and tested protocol.
Statues: Achieved. Sorta-kinda. Floating, beating EBs can be achieved within 15 days of aggregation in Lif- standard Media or 10 days with plating. But beating is uneven and unquantifiable.
hanging droplet method is very strenuous- and for some reason results in many drops not aggregating and cellualr debrees. But product is fairly even in size.
Plating results in very uneven and spreading pancake. Not sure is good for visualization and hypothesis testing without containment.
Microfluidics/platform
- prepare and test microfluidic chambers and microwells capable of trapping individual or aggregate ESCs and providing stable environment for differentiation.
Statues: have platform which sorta-kinda works on the short term. It CAN capture individual cells But I have yet to monitor aggregation and EB formation. Better, tested, platform seems to be avaliable from levenberg.
Roadblocks/shortcuts: 3-D printer, Levenberg.
Hypothesis testing
- prepare EB containing cells marked with markers forEctoderm, Endoderm, Mesoderm, CVP and CM.Prepare spatio-temporal map ofdifferentiation and analyze effect of EB size on parameters.
- Prepare EB containing cells marked with Wnt targets
- Prepare EBs containing cells with inducible expression of Wnt signals+ cells with marked Wnt targets.
Statues: no progress. i have the plasmid constructs. I can achieve succesful transfection. I thnik I can do selection. But I just can't move forward without clean cells to work with.
New six-month plan:????
Two week plan to 14.6:
Molecular/Cellular/EB differentiation:
31.5: prepare MEF feeders for R1 cells from Yechiel.
2.6: Seed the R1 cells on feeders.
4.6: split R1 cells>Feeders+gelatin
5.6/6.6: Transfection of R1 cells with Constituitive-CFP, E-Cadherin-Mcherry, Brachyury-GFP, Flk1-YFP, alpha-MHC Mcherry.
(Wendsday-Sunday) 7.6- 10.6: selection.
11.6 (Monday): SSC 96 well plating. try both needle and dilution plating.
14.6: measure fluoresecence in SSCs.
Microfluidics:
1.6: Talk to levenberg directly about blueprint.
2.6: Print microwell, H-wells and improved U traps on Architecture 3D printer.
4.6: Test 3D printer product
6.6 (If 3D does not work)- go into Nano room....
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