の効率的な胞子形成

The overall goal of this protocol is to sporulate budding yeast efficiently in a 96-well format. Although many large-scale screens have been carried out on the budding yeast, high-throughput studies looking at meiosis and sporulation have been limited. The technique described in this video has the advantage of sporulating yeast efficiently in a cost-effective manner in a 96-well format compatible with high-throughput screening.

I first got the idea for this method because I wanted to use a tiled two micron plasmid library in order to screen for high copy suppressors of a mutant that could not form refractile spores. Initially, my sporulation efficiencies were poor in the 96-well format. I found this was due to inadequate aeration.

Begin this procedure by thawing the SK1 yeast strain onto a yeast extract peptone glycerol or YPG plate. Use a sterile stick to scrape a small amount of cells out of the freezer vial containing the glycerol stock and spread it onto a YPG plate. Grow the yeast for 12 to 16 hours at 30 degrees Celsius.

Next, streak the yeast from the YPG plate onto a yeast extract peptone dextrose, or YPD, plate for single colones. Grow the yeast on the YPD plate for 24 to 48 hours at 30 degrees Celsius. After 24 to 48 hours of growth on YPD solid media, start a 25 milliliter YPD culture using a single colony from the YPD plate.

Grow this culture overnight in a 30 degree Celsius shaking incubator at 220 RPM until the cells reach an OD600 of 4.0 to 7.0. On the following day, use an appropriate amount of cells from the YPD culture to start a 125 milliliter culture in yeast extract peptone acetate, or YPA, with an OD600 of 0.1. Grow the culture in a 30 degree Celsius shaking incubator at 220 RPM until the OD600 is between 1.3 and 1.5.

Transfer the culture to centrifuge tubes and spin at 1800X G in a tabletop centrifuge for five minutes to pellet the cells. Wash the cells once with a 0.5 volume of sterile autoclaved deionized water. Spin again and discard the water.

Resuspend the cells in a 1X volume of sporulation media. To prepare cells of different genotypes, cells from thawed frozen stocks in a 96 multiwell plate are first transferred onto YPG solid media. Flame a 96 prong frogger and then cool it momentarily on a plate of solid media before touching it to the yeast colonies.

Grow the yeast overnight at 30 degrees Celsius. On the following day, transfer the cells from YPG solid media onto the YPD or selective solid media, depending on the genotype of the strain. YPD is used in this demonstration.

Grow the yeast at 30 degrees Celsius until colonies are formed for each strain. Aliquot 1.2 milliliters of YPD liquid media into each well of a 96 deep well plate. Transfer the cells from the solid media into liquid media.

Cover the plate with a plastic lid from a rectangular petri plate and hold the lid in place using a piece of tape to minimally restrict air circulation. Grow the cells overnight in a 30 degree Celsius shaker at 220 RPM. On the following day, centrifuge at 1800X G for five minutes.

Pour off the media and add 500 microliters of YPA to each well. And resuspend the pelleted cells. Cover the plate with a plastic lid held in place by tape.

Grow the cells for 15 hours in a 30 degree Celsius shaking incubator at 220 RPM. To provide proper aeration for efficient sporulation, add into each well of a 96-well plate either one three millimeter sterile solid glass bead or a sterile five millimeter by two millimeter magnetic stir bar. If all cells are of the same genotype, aliquat 500 microliters of cells plus media into each well of the 96-multiwell plate.

Cover the plate with a plastic lid held in place by tape. If cells are of different genotypes, spin the cells that are in YPA at 1800X G for five minutes and pour off the media. Add 500 microliters of sporulation media to each well.

And resuspend the pelleted cells in the sporulation media. Cover the plate with a plastic lid held in place by tape. If using glass beads, place the samples in a shaking incubator at 30 degrees Celsius and shake at 220 RPM for sporulation.

If using magnetic stir bars, place the samples on a stir plate inside of a 30 degrees Celsius incubator for sporulation. The exact stir speed for the magnetic bars is not very important, as long as all of the bars are moving energetically and the culture is aerated. 36 hours later, retrieve the samples to monitor progression through sporulation.

Withdraw six microliters of cells from each sporulating culture and add to 24 microliters of sporulation media in a 96-well coverslip plate. Leave the cells to settle for five minutes before examining them using an inverted microscope. A representative sporulation of two different strains is shown in these images of cells visualized using a 63X objective on an inverted microscope.

Refractile tetrads, indicated by the arrowheads, can be seen in the wild-type culture. But not in the culture containing the sporulation-deficient smk1 delete cells. Comparisons were made between sporulation efficiencies from cells sporulated in multi-well plates and cells sporulated using larger volumes in flasks.

The use of multi-well plates did not achieve the high efficiency routinely seen when using the more typical protocol of sporulating in flasks. Sporulating in multi-well plates with proper aeration can achieve sufficient sporulation efficiencies greater than 50%with the best results obtained using a five millimeter stir bar. However, given the high cost of the small stir bars, the efficiencies obtained using glass beads are comparable.

Although both five millimeter and seven millimeter stir bars fit within the well of a 96-multiwell plate, using a seven millimeter stir bar resulted in poor sporulation efficiency. The seven millimeter stir bar in a well tends to interact with a stir bar in an adjacent well. Preventing the bars from stirring properly and providing adequate aeration to the culture.

After watching this video, you should have a good understanding of how to efficiently sporulate yeast cells in liquid culture in the 96-well format. Once mastered, this process can be completed in six days, if done properly. This procedure can be used for screening different mutant strains, plasmids, or small molecule inhibitors for their effect on meiosis and sporulation.

After it's development, this technique was used to study the regulation of meiotic cell cycle and how cytokinesis and meiosis are coupled in sporulation in Saccharomyces cerevisiae.