Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

This study provides a standard protocol for studying the differentiation of Th17 cells, a subset of T helper cells that play a key role in immune responses. Through retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, scientists can study the particular function of the genes in regulating the Th17 differentiation. This study employs CRISPR-Cas9 technology for precise gene editing and utilized retroviral transduction to efficiently target specific genes in primary T cells.

Flow cytometry is used to evaluate transduction efficiency and monitor cell differentiation. In vitro Th17 polarizations are conducted to stimulate the differentiation of CT4+T cells. Our research aims to fill the gaps in understanding of how specific genes regulate Th17 differentiation.

We are using CRISPR-Cas9 along with retroviral transduction, which offers a faster and more cost-effective alternative to traditional knockout mouse models. Our protocol not only details the retroviral transduction of CD4 T cells, but also provides a comprehensive guide on our training sgRNA sequences and constructing plasmids, which is overlooked in other protocols. This paper outlines each step of the experimental process, ensuring that other researchers can easily replicate the methodology.

To begin, use the CRISPick tool to design the single guide RNA for ROR Gamma T knockout. Set the reference genome to Mouse GRCM 38, and specify the PAM sequence as NGG, based on the CRISPRko guide system. Enter the target gene name in the search box under the Quick Gene Lookup column.

Verify the system displays the correct gene ID and the full name of the target gene to ensure accurate selection. Choose one single guide RNA targeted on the RORC sequence based on the high combined rank and pick order to minimize off-target matches and increase on-target efficacy. Select the non-targeting single guide RNA sequence from the Mouse Gecko V2 libraries.

Amplify the single guide RORC and single guide non-target coding sequences using DNA polymerase in a PCR with primers designed for the single guide RNA and vector sequence regions. Clone the amplified sequences into the PMXU 6MCS vector, placing them between the U6 promoter and the guide RNA scaffold in frame with the mCherry fluorescent protein. Then set up a PCR program for vector construction.

One day before transfection, seed approximately eight times 10 to the power of six Plat-E cells in a 10 centimeter dish containing DMEM supplemented with 10%FBS, penicillin, and streptomycin. Incubate the cells at 37 degrees Celsius under 5%carbon dioxide until cells reach 80%confluency. One hour before transfection, replace the cell culture medium with eight milliliters of warm reduced serum medium without phenol red, containing 5%FBS, but no penicillin or streptomycin.

Prepare the transfection Mix 1, and Mix 2. Drop by drop, add Mix 2 to Mix 1, and mix gently. Incubate the mixture for 15 to 20 minutes at 20 to 25 degrees Celsius.

While gently swirling the plate, add the transfection mixture drop by drop to the Plat-E cells. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for six to twelve hours. After incubation, replace the medium with 10 milliliters of fresh cell culture medium and continue incubating at 37 degrees Celsius with 5%carbon dioxide for 48 hours.

Assess the transfection efficiency in Plat-E cells by detecting the retroviral vector tag using fluorescence microscopy. Next, centrifuge the virus-containing culture supernatant at 1, 500G for 10 minutes to remove cell fragments. Aliquot the virus-containing supernatant into one milliliter vials.

To begin, coat each well of a 24-well cell culture plate with two micrograms per milliliter of Anti CD3 Epsilon and four micrograms per milliliter of Anti CD28 in 500 microliters of PBS. Wrap the plate in Parafilm and incubate at four degrees Celsius overnight. After harvesting the mouse spleen, grind it in FACS buffer using sterilized ground glass to homogenize the tissue into a single cell suspension.

Filter the cell suspension through a 70 micrometer cell strainer into a 50 milliliter tube. Spin the splenic cell suspension at 800G for five minutes, and resuspend the pellet in two milliliters of red cell lysis buffer. Allow the mixture to sit at room temperature for five minutes.

Then add FACS buffer to reach a total volume of 15 milliliters. After centrifugation, resuspend the splenocytes in FACS buffer and filter them through a 40 micrometer cell strainer. Adjust the final volume to one milliliter with FACS buffer and isolate the CD4+T cells using commercial reagent kits, following the manufacturer's instructions.

Label the CD4+T cells with fluorescent antibodies mixture. After washing the stained cells with FACS buffer, isolate the naive CD4+T cells using a flow cell sorter. Now, after removing the plate-bound stimulation supernatant, rinse each well of a 24-well plate twice with 500 microliters of PBS.

Plate one times 10 to the power of six naive CD4+T cells in one milliliter of lymphocyte culture medium in each well and incubate for 18 to 24 hours. The next day, collect activated cells into 1.5 milliliter tubes, and centrifuge at 800G for five minutes. Resuspend the cell pellet in one milliliter of the infection mixture, and add one milliliter of the mixture into the wells of a 48-well plate.

Wrap the plate with Parafilm, and centrifuge at 800G for 90 minutes at 32 degrees Celsius, with acceleration and deceleration set at three. After centrifugation, remove the Parafilm and incubate the cells for four hours. To prepare antigen presenting cells, or APC, Resuspend the splenocytes in one milliliter of lymphocyte culture medium containing 50 micrograms per milliliter of mitomycin in a 15 milliliter tube.

Incubate splenocytes at 37 degrees Celsius with 5%carbon dioxide for one hour. Next, add PBS to the 15 milliliter mark and centrifuge at 800G for five minutes. Resuspend the splenocytes in one milliliter of lymphocyte culture medium, and count the cell number on a cell counter machine.

After retroviral infection, centrifuge the transduced CD4+T cells in 1.5 milliliter tubes, and resuspend the infected cells in 600 microliters of PBS. Place 0.5 times 10 to the power of six transduced CD4+T cells in one milliliter of lymphocyte culture medium with 1.5 times 10 to the power of six APC cells in a well of a 24-well cell culture plate. Supplement with two micrograms per milliliter of Anti CD3 Epsilon, two micrograms per milliliter of Anti CD28, and a Th17 differentiation cocktail.

Culture the cells for two days at 37 degrees Celsius with 5%carbon dioxide. After two days, centrifuge the cell suspension in 1.5 milliliter tubes and resuspend the pellet in one milliliter of lymphocyte culture medium containing three nanograms per milliliter of TGF Beta and 30 nanograms per milliliter of interleukin 6. Transfer the cells into the wells of a 24-well plate for two days.

Treat the differentiated cells with 50 nanograms per milliliter of PMA, 500 nanograms per milliliter of ionomycin, and five micrograms per milliliter of brefelden A in 500 microliters of lymphocyte culture medium in a new well of a 24-well plate. Incubate the cells at 37 degrees Celsius for four hours. Collect the treated cells into 1.5 milliliter tubes containing one milliliter of FACS buffer.

After centrifugation, stain the cells with PBS containing a fluorescent antibody mixture at four degrees Celsius for 30 minutes. Wash the stained cells with 300 microliters of PBS before fixing them with 300 microliters of 2%phosphate buffered formaldehyde at four degrees Celsius for 60 minutes. After washing the cells with PBS, resuspend them in 600 microliters of permeabilization buffer and centrifuge at 800G for five minutes.

Stain the cells with anti-interleukin 17A antibody in 100 microliters of permeabilization buffer for 60 minutes at room temperature. The retroviral infection efficiency, as indicated by mCherry positive cells, was approximately 40%Th17 differentiation efficiency was significantly reduced in cells treated with single guide RNA targeting the ROR Gamma T gene. The expression levels of RORC and interleukin 17A were substantially decreased in cells treated with single guide RNA RORC.