The overall goal of this procedure is to generate adult mice from induced pluripotent stem cells, or I PSCs by tetraploid embryo complementation. This is accomplished by first arriving. I PSCs from mouse embryonic fibroblasts using lentiviruses, carrying OCT four, SOX two, KLA four and cmic and supplementing the media with valproic acid or VPA.
Once the IPCs are generated, they are characterized by immuno staining, chromosome counting and embryo body formation. The next step is to generate recipient tetraploid blast assist via fusion and in vitro culture. Subsequently, the IPSC are injected into tetraploid blast assist and complemented blast assist are transplanted to recipient females for gestation.
In the final step, full term animals are delivered by cesarean section and to surrogate mothers. So postnatal development can be monitored. Ultimately if viable full-term animals are generated.
The assay IPSC line is considered fully pluripotent. The main advantage of this technique over other methods of generating I PSCs is that our method reproducibly generates fully pluripotent IPS cell lines IPS cell lines generated by these methods may be useful to other investigators wishing to compare the pluripotency of their IPS cells or to establish the equivalence of other reprogramming Methods. These methods will be useful for researchers wishing to generate fully pluripotent IPS cell lines for specific downstream applications such as in vitro differentiation assays.
Further the generation of all IPSC mice. Using telo complementation may serve as a useful means to determine the genetic and epigenetic stability of various cell lines. The microm manipulations involved in tetraploid embryo complementation are technically challenging.
It may take several months for someone to become proficient At the techniques IPSC is developed by this technique requires similar culture conditions as those used in traditional ESL gene targeting experiments. 24 hours after transfection of HEC 2 9 3 T cells with lentivirus. Remove the growth medium and replace it with 25 milliliters of fresh prewarm tech.
Medium return TRANSFECTED hex to the incubator for 24 hours, 48 hours after transfection of HEC 2 9 3 T cells with lentivirus collect growth medium containing lentiviral particles from the transfected cells. Remove particulate debris from the virus containing media by centrifugation at 3000 Gs for five minutes at four degrees Celsius. Next, concentrate the virus by ultracentrifugation through a 20%sucrose cushion for two hours at 112, 000 Gs at four degrees Celsius.
Carefully decant the into a waste container for decontamination without disturbing the viral pellet. Resuspend the viral pellet in 0.4 milliliters of mouse embryonic fibroblast medium and seal the tube. Then rock it gently at four degrees Celsius for 15 to 30 minutes.
Store a viral particles and single use aliquots at minus 80 degrees Celsius.IPSC. Lines are derived from mouse embryonic fibroblasts, or meth cells. Once IPSC colonies possess a bright refractive well-defined border and contain 30 to 50 cells, they are ready to be manually picked and expanded as independent IPSC lines.
Pick single colonies with a gel loading pipette tip and transfer each colony directly to a single well of a U bottom 96 well plate containing 20 microliters of 0.25%trypsin EDTA incubate picked colonies and trypsin for about eight minutes at 37 degrees Celsius or until the colonies dissociate into single cells. Monitor the progress of dissociation by light microscopy. Gentle tation may be necessary.
Transfer the dissociated cell suspension to a 96 well plate containing a feeder layer and 150 microliters of ESC medium supplemented with doxycycline and VPA When IPSC colonies reappear in each well split into larger wells for analysis and cell banking using standard ESC derivation and cell culture procedures to begin the procedure for generating tetraploid blast assist. One cell embryos collected from female mice are cultured to two cell embryos overnight in KSOM at 37 degrees Celsius, 5%carbon dioxide on the following day. Place A BTX micro slide in a 10 centimeter Petri dish.
Pour enough room temperature electro fusion media to submerge the slide in the solution, but not so much that the poles of the electrode are completely submerged. Switch on the BTX electro cell manipulator ECM 2001 and BTX enhancer 400. Connect the ECMs cables to the micro slides electrode and fix the cables to the side of the petri dish to prevent unintended movement of the slide.
Run one manual pulse to get a reading on the BTX enhancer and note the voltage of the a DC currents being applied. The AC current will control the speed at which the embryos align between the electrodes. The DC current will fuse the blasphemers and the pulse time will set the length of the DC pulse.
A good starting point is AC three volts DC 100 volts and times 0.05 milliseconds. The optimal DC varies in the range of 90 to 150 volts. Using a mouth pipette.
Take 30 to 42 cell embryos from KSOM culture and transfer them to a drop of electro fusion. Medium wash through several drops of electro fusion medium. Next, draw fresh electro fusion medium from the micro slide dish into the mouth pipette.
Take washed embryos and place them in the one millimeter gap between the electrodes on the micro slide. Be careful that the embryos are aligned down the middle of the gap and are not in contact with each other. Apply AC current by pressing the manual start button.
The embryos will rotate in the AC field until the plane of blasier contact is parallel to the electrodes. If embryos are not aligned in a few seconds, increase the AC setting. After embryos have aligned, press the manual start button again to apply the DC pulse with electro fusion medium in the pipette.
Collect the embryos from the micro slide wash embryos through several drops of KSOM medium and then culture embryos in KSOM under mineral oil at 37 degrees Celsius and 5%carbon dioxide. The blaster fusion should be completed in less than 30 minutes. In culture, monitor and select embryos with fused blastomeres successfully.
Fused embryos will appear to be in the one cell stage discard lysed and two cell embryos after 30 minutes in culture, continue to culture fused embryos in micro drops of KSOM under mineral oil at 37 degrees Celsius. 5%carbon dioxide to prepare IPCs for blast injection thaw previously banked IPCs and plate them on feeders in ESC medium passage to the cells at least once on feeders. After thawing before use for injection.
One well of a six well plate containing 70 to 80%confluent IPCs will provide more than a sufficient number of cells for injection aspirate growth medium and wash the cells with three milliliters of PBS without calcium or magnesium at 0.5 milliliters of prewarm, 0.05%tripsin EDTA to the cells and incubate at 37 degrees Celsius for 10 minutes with occasional rocking at 3.0 milliliters of ESC medium and iterate to obtain a single cell suspension. Observe cells by light microscopy to ensure a single cell suspension, colonies or cell aggregates will clog the injection pipette. Once a single cell suspension has been achieved, add one milliliter of ESC medium to the well and return the plate to the 37 degrees Celsius incubator.
Incubate for about 15 minutes or until the majority of feeder cells have begun to adhere, gently remove the medium containing the IPCs. Taking care not to dislodge the weekly adherent feeder cells. Place the IPCs in a 15 milliliter conical tube containing five milliliters of ESC medium centrifuge at 200 Gs for five minutes.
Aspirate the supernatant and remove the remainder of ESC medium with a micro pipette. Tap the tube to dislodge the pellet and gently resuspend the cells in 0.2 to 0.5 milliliters of pre chilled FHM medium. Store the cells on ice until and during injection into tetraploid Blast assists For the protocol for blast assist injection, please refer to the manuscript accompanying this article and to a previously published strove article, an excerpt of which is being shown on the screen here.
Examples of the morphological progression from fibroblast to IPSC colonies during the course of a reprogramming experiment are shown in these images. Morphological heterogeneity and partially reprogrammed IPSC colony formation should be observed starting at four to five days after doxycycline VPA edition as shown in panels B to EESC. Like IPSC colonies should appear between seven to 10 days as shown in Panel F.The production of one cell telo embryos by electro fusion of diploid two cell embryos is highly efficient.
It is routine to observe up to 95%of treated two cell embryos successfully fused to produce tetraploid one cell embryos. The protocol used in this experiment for injecting IPSC into tetraploid blast assist is similar to the protocol for injection of E SES into diploid blast assist to generate chimeric mice to easily rule out diploid chimera generation, the pigmentation of the mouse strains used to generate IPSes and tetraploid embryos are carefully chosen when possible. In this example, tetraploid embryos are produced from an albino strain and IPSC are generated from a pigmented strain.
Thus newborn IPSC mice display pigmented eyes and a uniformly pigmented coat color in adulthood, whereas diploid kyras would display mixed albino and pigmented coloring. The number of live pups born depends on the cell line as shown in this table. For an average good IPSC line, experimenters should expect between 1%and 10%of injected blast assist to yield live newborn pups.
After watching this video, you should have a good understanding of how to derive fully pluripotent IPS cells and also how to validate their developmental potential. Using the tetraploid complementation assay Once mastered this technique from derivation of MES to newborn IPSC, mice can be completed from two to three Months. Development and maintenance of I PSCs require a high level of cell culture technique.
Previous experience with ESL culture is essential. Don't forget working with lentivirus, especially concentrated lentivirus can be extremely hazardous. One should always wear the personal protection equipment required by your institution and be sure to treat everything that's come in contact with the virus, with 10%bleach for at least 20 minutes Following this procedure.
All IPSC mice and their tissues can be analyzed for physiological, pathological and behavioral phenotypes.
