トランスフェクション、選択、およびAAVS1セーフハーバーへのGFP遺伝子とTALENを標的とする人間の人工多能性幹細胞のコロニーピッキング

The overall goal of this procedure is to generate an isolate AA vs one safe harbor, targeted inducible pluripotent stem cell or IPSC clones expressing GFP. This is accomplished by first delivering a AAVs one talons and a A VS one C-A-G-E-G-F-P donor plasmids efficiently into IPCs by nucleo affection. In the second step, the A a vs one targeted cells are selected by pur mycin on mouse embryonic feeder cells.

Next, the individual GFP expressing A A VS one targeted clones are picked into a matrigel coated 96 well plate. In the final step, the A A VS one G-F-P-I-P-S-C clones are expanded with E eight medium and standard IPSC culture techniques. Ultimately, immunofluorescence microscopy and flow cytometry are used to show the uniform and robust expression of GFP in AA VS one targeted human IPSC clones.

Visual demonstration of this method is critical as the colony picking steps involve micro dissection of targeted clones using special colony picking tools under a microscope, which is difficult to learn during side-by-side instruction. Begin by washing the IPSC cultures one time each with PBS. Then add one milliliter of 37 degrees Celsius gentle cell dissociation reagent to each well and incubate the cells at 37 degrees Celsius for approximately five minutes when greater than 50%of the cells have dissociated from the culture vessel.

Use a P 1000 pipette to mechanically disrupt any remaining cell clumps or attached cells. Then add two milliliters of E eight medium to each well and use a 10 milliliter pipette to further disaggregate the cultures into single cell suspensions. Now pool the harvested cells into a 15 milliliter conical tube and spin them down.

Re suspend the pellet in a minimal amount of E eight medium for counting. Then after confirming the viability of the cultures by trian blue exclusion, as well as their sufficient dissociation transferred 3 million cells into each of two 15 milliliter conical tubes while the cells of centrifuging set the electroporation system to the cell type specific program for the human embryonic stem cell line H nine. Then add 10 micrograms of the homologous recombination donor alone to one pellet for the control sample and 10 micrograms of the homologous recombination donor plasmid, along with five micrograms of each talon plasmid for the experimental sample.

Next, add 100 microliters of room temperature complete P three primary cell transfection solution to each of the control and experimental pellets and resuspend the cells. Transfer the samples into individual cuvettes and then ate the samples immediately after the transfection. Add 500 microliters of room temperature E eight medium to each Q vet and then transfer the transfected IPSC samples dropwise into individual 10 centimeter dishes containing DR four mouse embryonic fibroblasts Before picking the colonies, co-teach well of a 96 well plate with 50 microliters of basement membrane matrix solution and then store the plate overnight at four degrees Celsius the next morning.

When the plate has reached room temperature, aspirate the excess solution and load each well with 100 microliters of E eight medium supplemented with 10 micromolar Y 2 7 6 3 2. Next, place an inverted microscope into a biological safety cabinet and sterilize it with a light spray of 70%ethanol. Then place a glass pasture pipette over a bunsen burner to create a colony picking tool with a tiny hook at the tip after sterilization with 70%ethanol.

Place the hook in the hood to dry and then place the IPSE culture on the microscope stage to pick the clones. Locate a suitable colony under a low magnification and use the colony picking tool to trace and quarter it. Then gently scrape the quartered colony from the culture vessel and use a P 200 pipette set to approximately 30 microliters to collect the freely floating IPCs plating the cells directly onto the basement membrane matrix.

As they are harvested, the IPCs should attach within two to four hours of plating. Finally incubate the picked colonies overnight at 37 degrees Celsius and refresh the cells with complete E eight medium the following morning. It is important to transfect only high quality I PSCs throughout the experiment.

The IPSC cultures should contain mainly distinct colonies bearing a cobblestone like morphology as the cells seen here and the differentiated cells should not more than 10%of the culture, the transfect ability of the IPCs can be assessed and optimized using the small PAX GFP vector as transfection with PAX GFP typically represents the maximum achievable efficiency due to its small size. The transient expression of a A VS one C-A-G-E-G-F-P intel and transfected IPCs peaks at 48 to 72 hours post transfection, which can be confirmed with a small portion of transfected cells by flow cytometry. The transfection efficiency can vary greatly.

Even in experiments with A GFP positive fraction is only 10%Individual clones displaying a uniform fluorescence should be large enough for colony picking after pure mycin selection. Further, once in the 96, well format the expanded targeted IPSC clones should exhibit a stable and uniform expression of GFP. After watching this video, you should have a good understanding of how to transfect I PSCs efficiently by nuclear affection and how to isolate AV one safe harbor.

Targeted IPSE clones using colony picking tools.