Lineage Labeling & Cell Transfer

Reagents and Equipment:

1x Modified Holtfreter's buffer:      60mM NaCl
                                                            0.6mM KCl

                                                            0.9mM CaCl

                                                            5mM HEPES (pH 7.4)

Prepare 10x stock solution.

Antibiotics:

For cell transplantation, use full-strength (100x) stock solution of Penicillin (5,000 units/ml) - Streptomycin (5,000 ug/ml) (TermoFisher 15070063).

Dextran Dye Solution:

1. Dissolve 1mg Dye (lysine-fixable dextran conjugated to rhodamine and biotin, 10,000 MW) in 40ul of 100mM KCl.
2. Warm up to ~40oC briefly to dissolve visible clumps.
3. Spin in spin-X column (0.45um- Costar #8162) for 2 minutes in microfuge.
4. Remove upper filter from the column, add 1.2ul green food coloring (final concentration ~3%).
5. Wrap tube with foil, store tube at 4oC. (Dextran is photosensitive.)
6. Warm briefly to 40oC to ensure that all dye is in solution just prior to use. Vortex the tube before use.

Capillaries for puller: FHC # 30 -30 - 0

Filaments for puller: Sutter # FT 320B

Sutter Instruments Needle Puller: Model P-87

Program Settings for Puller: 295 - 20 - 200 – 40

Microinjector: Narishige IM-5A/B with 3cc syrninge.

Procedure:

1. Warm dye solution, Hotfretter’s buffer and antibiotic solution in warm (40°C) water before collecting eggs.  Add 1/10th volume of 10x Holtfretter’s buffer to full- strength Pen-Strep solution just before use.

2. Inject dye into donor eggs at 1-cell stage with glass needle. Can inject with intact chorions.
3. Dechorionate donor and host embryos, allow to develop to oblong-sphere stage (3.5-4.0 hpf at 28.5°C).
4. Place embryos in 9-well glass plate with Holtfretter’s buffer + Pen-Strep.
5. For cell transplantation, break off a clean injection needle closer to shank to yield larger bore size, roughly 1.5-2 cell diameters across.
6. Using Narishige microinjector, withdraw a column of cells from a labeled donor embryo. Ideally cells should form a coherent column inside the needle rather than a loose suspension. Insert needle into unlabeled host embryo parallel to the yolk boundary, a third of the distance from the boundary to the animal pole, and slowly extrude column of donor cells. Transfer embryos to petri dish with embryo-rearing water and incubate at 28°C to the desired stage.