分裂酵母の交配 mating

Aim

分裂酵母の１倍体細胞をかけ合わせて、減数分裂-胞子形成を行わせ、子孫の１倍体細胞から目的の遺伝子型、表現型を持つ細胞を取得する。

Materials

Glusurase

2 μL for 100 μL spore suspention (see below)

discontinued

alternatives

1 μL for 30 μL spore suspention (see below)

SPA or SSA plate

See medium recepies

100% Ethanol

Selective plates

Methods

Spore formation

Grow cells of opposite mating type (h+ and h-) that are to be closed.

e.g. 2 mL YEA culuture to 1.0x10^7 cells /mL

h90 strains can be used if resultant offsprings can be efficiently selected by antibiotics or some other selection methods.

Collect cells by centrifiguration (~3000 rpm, 1 min).

Remove media, and wash cell pellet with 500 μL DDW.

Cell pellet is suspended into rouglhy same amount of DDW.

Mix the same amount of cell suspention of opposite mating type.

if h90 is crossed to h- or h+, mixiing ratio should be 1:3.

Spot 10 μL of cell suspention mixture onto SPA plate.

Wait for several minuites until the spot becomes dry up

2-3 days 25-30℃ incubation (Higher temperature like 32℃ decreases the mating efficiency).

Random Spore

Check sporulation by microscope.

Take a half of SPA spot by toothpick and suspend it into 100 μL DDW containing 2 uL of Glusurase. Incubate 1h.

Using 0.5 μL Glusurase, incubation can be extended to 4 h ~ overnight.

34℃ is better for incubation.

Rotation by tube rotatoe is better than settling.

(Optional) Add 43 uL 100% EtOH after the incunation and wait 15 min.

This step kills vesitative cells and non sporulated diploids completely. If you mind the survivor of non-mating cells, this option might give better results.

S.pombe spore sacs is naturally degraded by longer incubation on SPA. Therefore, only an EtOH treatement inteased of glusurase treatment mighth be sufficient for random spore of S. pombe.

Add 900 μL DDW, and dilute for suitable spore concentration. Plate diluted spores onto selection plates.

e.g. If mating efficificency ~90% and crossing result is expected to follow Mendelian, 10 μL of suspention is suitable. So, plate 100 uL of 1:10 dilution onto selection plate (10 cm).

Tetrad analysis

Follow the manufacture's instruction.

Remarks