Gene targeting mutagenesi casuale per selezionare eterocromatina-destabilizzare Proteasome mutanti in lievito di fissione

The overall goal of this random mutagenesis is to screen for mutations that destabilize heterochromatin. This method can help answer key questions in the heterochromatin field, such as factors that regulate the formation and maintenance of heterochromatin. The main advantage of this technique is that it can specifically target a desired gene for mutagenesis, even if the gene is essential.

To begin, prepare yeast extract with supplements, or YES, without adenine, Pombe Glutamate Medium, or PMG, and PMG without adenine, by mixing the components as shown in this table. After autoclaving and adding G418 if necessary, stir the medium for another five to ten minutes. Then, aliquot the media into 90 milliliter Petri dishes and store the plates at four degrees Celsius.

Using the cloned rpt4 positive, with its five prime and three prime UTRs and silent mutation, as well as primers p5 and p6 and a special polymerase, designed to generate a high error rate, perform error-prone PCR in a total volume of 50 microliters. After generating a transformation-fusion construct, according to the text protocol, inoculate ten milliliters of YES medium with yeast, and incubate it for more than 16 hours to saturation. Use 200 milliliters of YES medium to dilute the cells to an OD600 of 0.2, and incubate the culture at 30 degrees Celsius with shaking for five to six hours to an OD600 of 0.6 to 0.8.

Calculate the volume of cells that add up to OD600 of 30. Then, dispense the cells into four 50 milliliter conical tubes and chill them on ice for 10 minutes. Harvest the cells by centrifugation at 1050 G and four degrees Celsius for three minutes.

Then, place the microfuge tubes with cassette DNA and 10 electro cuvettes on ice. While still on ice, discard the supernatant and add 15 milliliters of 1.2-molar sorbitol, gently shake the tubes to re-suspend the cells, then centrifuge the cells at 1050 G and four degrees Celsius for three minutes. Discard the supernatant and perform a second sorbitol wash.

After centrifugation, discard the supernatant. Then, re-suspend the cells and collect them all in one 15-milliliter conical tube. Next, add 1.2 molar sorbitol up to 2.4 milliliters.

Keep the tube of cells on ice. Then, add 200 microliters of sorbitol-suspended cells to a tube containing the fusion PCR construct, mix well, and transfer the sample to an electro cuvette. It is important not to use an excess amount of PCR cassette, as it will give discharge errors.

Electroporate the cells using the following options:add 600 microliters of 1.2-molar sorbitol to each electro cuvette for a total volume of 800 microliters. Then, spread the cells onto four YES plates. Incubate the plates at 30 degrees Celsius for 24 hours.

Then, perform replica plating on YES plus G418 before incubating the plates for an additional three days. For each YES plus G418 plate, perform replica plating to YES without adenine and PMG without adenine plates. Incubate the replica plates at 30 degrees Celsius for one to two days until some of the colonies on the YES without adenine plates show a white coloration.

Compare YES without adenine and PMG without adenine plates and select cells that show pink or white on the YES without adenine plate, and also grow on the PMG without adenine plate. Do not select colonies without growth on PMG without adenine, as they are false positives. Pick each colony and add it to 10 microliters of SPZ solution containing 2.5 milligrams per milliliter of zymolyase 100T.

Then, incubate the colonies at 37 degrees Celsius for at least 30 minutes. Following the incubation, use one microliter of this solution as the starting template for colony PCR. After digesting selected colonies and carrying out gel electrophoresis according to the text protocol, mark the colonies whose PCR products were cut by XHO1 and patch them to YES plus G418 plates.

Following the isolation of gDNA from fission yeast cells, and sequencing of the PCR products according to the text protocol, compare the obtained sequence with the wild-type sequence to identify mutations. Once the mutations are re-introduced into wild-type cells, use spotting to confirm the phenotype in the newly-made mutant cells. The rpt4 mutants generated using the protocol demonstrated in this video can be analyzed by assessing the colors of the colonies.

As shown in this figure, the colonies were spotted onto the relevant plates in decreasing cell number. The adenine reporter inserted in the heterochromatin region is silenced in wild-type cells and produces red colonies on YES without adenine plates. Once the heterochromatin is destabilized and the adenine reporter is expressed, white colonies can be observed on the YES without adenine plates as seen with the clr4-delta mutant.

The screened rpt4 mutants are as shown with the rpt4-1 mutant exhibiting the most severe heterochromatin destabilization. Once mastered, this technique can be done in two weeks to get the desired mutants. While attempting this procedure, it’s important to eliminate the false positives as they are more frequent than expected.

Following this procedure, other methods like protein purification and enzyme activity tests can be performed in order to answer additional questions, like the nature of the proteins. After watching this video, you should have a good understanding of how to generate mutants in a target gene with the desired phenotype.