Approccio genetico in avanti per scoprire la resistenza allo stress geni nei topi - Uno Schermo high-throughput in cellule ES

The overall goals of this method are to efficiently select for stress resistant mutant embryonic stem cells rapidly identify associated genes and cost-effectively generate viable mouse models. This methods can help answer key questions in the aging field, allowing us to identify genes involved in stress resistant and the enhancement of health spans and lifespan in mammals. So one advantage of this technique is that it allows for the cost effective selection of novel mutants and for the generation of phenotypic mouse models following embryonic stem cell screening as a surrogate model.

To begin culturing ES cells first thaw one vial of mitomycin C activated primary mouse embryonic fibroblasts or P MFS in a 37 degree Celsius water bath to be used as feeder cells for the ES cell culture. Transfer the cells into five milliliters of E Es cell medium and centrifuge them at 100 times G for five minutes. Aspirate the supernatant and resuspend the cells in 30 milliliters of ES cell medium.

Distribute the cells to two 100 millimeter plates and to two T 25 flasks. Incubate the cells at 37 degrees Celsius and 5%CO2 for 24 hours. Next, thaw one vial of C nine ES cells in a 37 degrees Celsius water bath.

Transfer the cells into five milliliters of ES cell medium and centrifuge the tube at 100 times G for five minutes. Aspirate the S supernatant and resuspend the cells in five milliliters of ES cell medium. Then plate them into one of the T 25 flasks containing feeder cells.

Incubate at 37 degrees Celsius and 5%CO2. Replace the medium daily. After two days, the ES cells should be approximately 80%confluent to passage.

These cells wash them once with five milliliters of PBS and add 2.5 milliliters of 0.25%trypsin EDTA incubate at 37 degrees Celsius for 10 minutes. Now add 2.5 milliliters of ES cell medium and pipette up and down 15 times to break up cell clumps. Transfer the cells to a 15 milliliter conical tube and centrifuge at 100 times G for five minutes.

Aspirate the supernatant and resuspend the cell pellet in four milliliters of ES cell medium. Then transfer one milliliter of the cells to each of two 100 millimeter plates containing nine milliliters of ES cell medium. Incubate the plates in a cell culture incubator and replace the medium daily to prepare 96 well feeder plates for library construction.

Thaw three vials of mitomycin C inactivated P, mfs, and resuspend each vial in 33.3 milliliters of ESL medium. Combine the cells to yield a 100 milliliter cell suspension from this plate. 100 microliters of cells per well into 10 96 well plates 24 hours later.

Trypsin eyes the C nine ES cells growing on the 100 millimeter plates. After counting, prepare six 15 milliliter tubes each containing five times 10 to the sixth ES cells and resuspend them in 0.8 milliliters of PBS. To generate the ESL mutant library, prepare six electroporation cuvettes by adding the 0.8 milliliters of cells.

One microgram of the piggyback or P-B-U-P-A vector and 20 micrograms of the MPB transposes mixed gently by pipetting and place the cuvettes on ice. Perform electroporation using the exponential setting at 250 volts, 500 microfarads and infinity ohms. Pool the transfected cells in a T 75 flask containing 98 milliliters of ESL medium.

Mix the cells by gentle pipetting and transfer 50 milliliters to a reservoir. Use a 12 channel pipette to transfer 100 microliters of cells to each well of the 96 well feeder plates incubate at 37 degrees Celsius and 5%CO2 after 24 hours. Feed the cells daily with fresh ESL medium containing 150 micrograms per milliliter G four 18 to begin replication of the ESL library.

First prepare new 96 well feeder plates and gelatin coated plates as indicated in the text protocol. Wash the ESL library plates one time with PBS and add 50 microliters of 0.25%trypsin EDTA After incubating the cells at 37 degrees Celsius for 10 minutes, add 50 microliters of ESL medium and pipette up and down. Transfer 50 microliters of this cell suspension from the original 96 well plate to the corresponding row of a 96 well feeder plate known as the master, which already contains 150 microliters of E es cell medium.

Then transfer the remaining 50 microliters of the cell suspension from the original 96 well plate to the corresponding row of a gelatinized 96 well plate known as the replica, which already contains 150 microliters of ESL medium. Incubate both sets of plates at 37 degrees Celsius and 5%CO2 overnight after 24 hours. Replace the medium with ESL medium containing 150 micrograms per milliliter of G four 18 and change the medium daily to freeze the master plates.

Wash and tryps anize the cells and then add 50 microliters of two x freezing medium pipette up and down 10 times to mix. Put the 96 well plates into a tightly sealed styrofoam box and freeze at minus 80 degrees Celsius for up to four months to generate plates for later genomic DNA isolation. First prepare 10 each of 96 well feeder cell and gelatinized plates.

Then wash and tryps anize the cells in the 96 well replica plates add 50 microliters of ES cell, medium to each well and pipette to break up cell clumps. Transfer 50 microliters of this cell suspension to the corresponding rows of the gelatinized DNA plates. Transfer the remaining 50 microliters to a reservoir containing five milliliters of ESL medium.

Next, transfer the entire volume of cells from the reservoir to 100 millimeter feeder plate to freeze the 96 well DNA plates grow cells to 80 to 90%co fluency and wash the cells twice with PBS. Store the plates at minus 20 degrees Celsius to generate homozygous mutants. Transfer five times 10 to the six cells from each of the sub library plates onto a gelatinized 100 millimeter plate.

Add ES cell medium containing one microgram per milliliter of doxycycline to a final volume of 10 milliliters. Incubate the plates at 37 degrees Celsius and 5%CO2 following doxycycline selection seed 6.6 times 10 to the six cells onto a gelatinized 150 millimeter plate in 30 milliliters of ES cell medium containing 7.5%heat inactivated FBS and 10 micromolar paraquat incubate the cells at 37 degrees Celsius and 5%CO2 for seven days. Replace the medium with fresh ES cell medium after seven days of stress selection once colonies have grown.

Prepare 96 well feeder plates containing 100 microliters of fresh ESL medium on the day of picking prepare ubo. 96 well plates with 50 microliters of 0.25%tripsin EDTA. Remove the medium from the 150 millimeters stress selection plate and replace with PBS using a micro pipette set to two microliters.

Pick individual colonies into each well of the 96 well you bottom plate after all the colonies are picked, incubate the plate at 37 degrees Celsius and 5%CO2 for 10 minutes. After the 10 minute incubation, add 50 microliters of ESL medium per well and pipet up and down to break up the cells. Transfer 100 microliters of this cell suspension to the corresponding row in the 96 well feeder plate culture.

The cells at 37 degrees Celsius and 5%CO2 overnight and replace the medium daily. Once cells have grown to 80 to 90%co fluency aspirate the medium, wash the cells with PBS and trypsin eyes using 50 microliters of 0.25%trypsin EDTA after a 10 minute incubation at 37 degrees Celsius. Add 150 microliters of fresh ES cell medium and transfer 100 microliters each to the two replica gelatinized.

96 well plates when these replica plates reach 80 to 90%co fluency freeze one set of plates with two x freezing medium to expand stress resistant colonies for genomic DNA isolation wash and tryps anize the cells from the other set of plates. Transfer the cell suspension from one well of the 96 well plate to one well in a gelatinized 24 well plate culture. These cells in 0.5 milliliters of ESL medium until they reach 80 to 90%co fluency, beginning with three times 10 to the seventh esl.

The gene entrapment process described in this video demonstrates approximately 0.04%efficiency with 22, 440 independent mutants being generated. Of these mutants, 17 or 0.08%were stress resistant. As measured by resistance to paraquat.

Three paraquat resistant clones were selected for further characterization. Pig L TM one and ROF fillin mutants cells with ROF fillin mutation were able to produce homozygous mutant progeny while pig L and TM one were not. Stress resistance to paraquat as characterized by the percentage of surviving cells compared to the parental embryonic stem cells was increased for all three mutants.

Following removal of the piggyback transpose on these cell lines, lost stress resistance following introduction of mutant cells into blast assist for mouse production. Skin fibroblasts isolated from the resulting pig L mutant mice showed reduced levels of endogenous reactive oxygen species or ROS as well as maintain stress resistance to paraquat Once mastered, you can identify stress resistant embryonic stem cell clones in five weeks by following this protocol. Following this procedure, other selections can be performed using different compounds in order to answer additional questions surrounding the genetic basis for a variety of stress resistant phenotypes.

After watching this video, you should have a good understanding of how to generate mutagen embryonic stem cell libraries and to screen for stress resistance mutants. The use of the replica plating strategy is particularly crucial for downstream mouse production.