The overall goal of this structure-function method is to dissect at the molecular level the role of different protein domains, or disease-relevance mutations, in naive or differentiated mouse embryonic stem cells. This method can help to reveal how different residues or domains of a protein can contribute to its function in a given cellular context. The main advantage of this technique is that it allows very efficient targeting of rescue constructs to the genome of knock out embryonic stem cells, or ES cells, and the investigation of their role in different cell types.
To achieve this, we combine three highly efficient technologies. These include improved mouse embryonic stem cell isolation, recombination-based targeting factor assembly, and ES cell targeting via recombination-mediated cassette exchange, or RMC. Demonstrating some of the procedures will be Jinke D'Hont, a technician from my laboratory.
Recombination-mediated cassette exchange, or RMCE, allows the interchange of DNA fragments between a vector and a genomic locus. RMCE takes advantage of hetero-specific recombination sites that do not cross-react and that are embedded in a genomic locus. In the presence of a donor plasmid that contains a DNA fragment flanked by the same hetero-specific sites, the recombinase will insert this DNA fragment into the RMCE-compatible genomic locus because of double simultaneous translocation.
Only upon correct recombination, the trapped promoterless Neomycin-resistance gene in the Rosa 26 docking site, will be restored via a PGK promoter in the incoming targeting vector, which renders drug-resistance. This Neomycin resistance trap system results in a very high targeting efficiency, often close to 100%The day before blastocyst isolation, add 0.1%gelatin, to 12 well-cultured plates. And incubate them at 37 degrees Celsius for five minutes.
Aspirate the gelatin solution. Then seed one quarter of a vial of P2 Mouse Embryonic Fibroblasts, or MEFs, into MEF medium, and divide over the 12-well plate. Incubate the 12-well plates of MEFs in two milliliters of MEF medium at 37 degrees Celsius.
And grow the cells to a confluent monolayer. Then add 10 micrograms per milliliter of Mitomycin seed to the wells. And incubate the cultures for three hours at 37 degrees Celsius, to inactivate the cells.
After selecting heterozygous knock out mice containing an RMCE cassette in the Rosa 26 locus according to the text protocol, set up matings to breed RMCE-compatible heterozygous knock out mice, with heterozygous knock out mice. The following morning, check for copulation plugs by lifting the female by the base of the tale, and examine the vaginal opening for a whitish mass. If the plug is difficult to see, with an angled probe, slightly spread the lips of the vulva, then separate the plugged females from their males.
To collect blastocysts at 3.5 days post-coitus, or DPC. After euthanizing the pregnant females, make a mid-ventral incision, and use fine scissors and forceps to dissect the uterus and oviduct. Next, bend a 26-gauge needle into a 45-degree angle attached to a 1 milliliter syringe filled with M2 medium.
And insert the needle into the end of the uterus that is closest to the oviduct. Use fine forceps to hold the needle in place while pushing the plunger to flush the blastocysts from the uterus into the lid of a 10 centimeter dish. The uterus will swell, if the flushing is successful.
Use a mouth pipette to collect all the embryos in a drop of M2 medium, and wash the embryos twice in a drop of M2 medium. Immediately after washing the embryos, use PBS, to wash the mitomycin-C inactivated cells twice. Then using a mouth pipette, plate each blastocyst onto a separate well of the mitomycin-C treated MEFs, in SRES cell medium, supplemented with pluripotent or 2I.
Incubate the cultures at 37 degrees Celsius, and 5%carbon dioxide. Refresh the SREF cell medium every two to three days. Examine each blastocyst under a stereo microscope at 4X magnification, and check for hatching and detachment to the MEF layer.
After 10 to 12 days of culture, under a stereo microscope, use a P10 pipette with disposable tips, to pick individual icium outgrowths. Transfer each outgrowth into approximately 10 microliters of medium to a V-shaped 96-well plate, containing 30 microliters per well of PBS. Using a multi-channel pipette, add 50 microliters of 0.25%trypsin to each well.
And incubate the plate at 37 degrees Celsius and 5%carbon dioxide for three minutes. Add 100 microliters of pre-incubated FPS-containing ES cell medium, and dissociate the icium outgrowths into single cells by pipetting 10 to 15 times. Then transfer the dissociated cells to mitomycin-C treated 96 well MEF plates.
The following day, change the SR-based medium. Expand the ES cells in a similar fashion from 24 to 6-well plates. After identifying rescued CDNA vectors according to the text protocol, prepare a 10 microliter LR reaction using 100 nanograms of rescue CDNA vector, 150 nanograms of pre-excised RMCEDV1 vector, and two microliters of recombinase mix, containing a phage-encoded integrase, an excisionase, and a bacterial integration host factor.
Incubate the reaction at 25 degrees Celsius for two hours. To transform the recombined vectors, add 5 microliters of each mixture, to 40 microliters of heat shock competent E.Coli bacteria, in a 2 milliliter skirted screw cap tube. And incubate the sample on ice for 20 minutes.
Then incubate the cells at 37 degrees Celsius for five minutes. Add one milliliter of LB medium to the tube, and incubate the cells at 37 degrees Celsius for one hour. Plate 50 microliters on agar plates with ampicillin.
And grow at 37 degrees Celsius overnight. Identify colonies with the correct targeting vector by using a P200 tip to randomly pick five colonies. Transfer the tip to a glass test tube containing two to five milliliters of LB medium, and grow overnight at 37 degrees Celsius.
After extracting the DNA from each colony, validate the samples by digesting 0.5 to two micrograms of DNA, and separate the samples on a 1%agarose gel. Start a culture of RMCE compatible knock out ES cells, and passage them at least twice on MEFs, in FBS-based ES cell medium. Then split the ES cells on a gelatinized six-well plate.
The following day, use 1.5 milliliters of FBS-based ES cell medium to refresh the ES cells that are about 50%confluent. Combine one microgram of pre-excised RMCEDV1 targeting vector, containing rescue CDNA, and one microgram of FLIPe expression plasmid, in 250 microliters of pure DMEM medium. Add seven microliters of lipofectin-based transvection reagent to 250 microliters of pure DMEM medium.
And incubate the solution for five minutes at room temperature. Combine the DNA and lipofectin solutions. And incubate the mixture at room temperature for 20 minutes.
Then pipette the transvection mixture onto the refreshed ES cells, and gently swirl. One day after transvection, split all ES cells from the tube to a 10-centimeter culture dish, with a confluent layer of DR4 MEFS, and 10 milliliters of FPS-based ES cell medium. Two days after transvection, select ES cell clones with the correct FLIP-e mediated cassette exchange, by adding G418 to the medium.
As shown here, massive killing of non-targeted colonies should be visible after three to five days. Colonies should appear after seven to 10 days. Pick these colonies, then expand them, and verify the clones as demonstrated earlier in this video.
To differentiate ES cells into embryoid bodies, or EBs, after culturing knock out ES cells with Rosa 26-driven rescue constructs according to the text protocol, allow EBs to form in the non-adherent dishes for 30 days. Refresh the medium every two to three days by transferring the EB suspension to a 50 milliliter tube. And let the EBs settle by gravity.
Remove the supernatant, add fresh medium, and transfer the EB suspension to a bacterial-grade dish. Analyze the ES cells and EBs by immunofluorescence and TEM, according to the text protocol. Using the structure function method, five pre-excised RMCEDV1 targeting vectors were generated with an efficiency of 100%and these rescue constructs were targeted via RMCE, to P120 catenin knock out ES cells, with an efficiency of 97%Cystic EB morphology can be used as a phenotypic readout to screen different rescue constructs.
As proof of concept, R26-driven P120 catenin isoform 1A rescued the p120 catenin knock out phenotype. To test whether E-cadherin independent membrane anchoring of P120 catenin enabled csytic EB formation, the k-ras membrane targeting motif, CAAX, was fused to the carboxy terminus of P120 catenin, and introduced via RMCE, to P120 catenin knock out ES cells, before EBs were made from them. However, this construct serves a dominant negative that does not allow binding and stabilization of E-cadherin.
And as a consequence, does not rescue the p120 catenin knock out phenotype. Epithelial-mesenchymal transition, or EMT, is an essential developmental process that also occurs during ES cell differentiation. When expressed in p120 catenin knock out ES cells, the EMT inducers, ZEB1, ZEB2, or snail that can directly repress various epithelial marker genes like E-cadherin, failed to restore cystic EB formation.
Once mastered, RMCE compatible ES cells can be isolated within one month. RMCE compatible targeting vectors can be generated within one week, and targeted rescue of ES cells can be achieved within one month using this technique, if it is performed properly. After watching this video, you should have a good understanding of how to use RMCE to perform structure function studies in naive or differentiated embryonic stem cells.
