Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Handling of lentivirus is performed as per the regulation of the Department of Environmental Health and Safety of National Taiwan University College of Medicine.

1. Preparation of immortalized hematopoietic stem and progenitor cell lines (iHSPCs)

1. Maintain iHSPC cell line in complete RPMI 1640 medium containing 100 ng/mL FL and 1 µM β-estradiol.
2. Passage the cells at a ratio of 1:10 every 3 days.
   ​NOTE: Make complete RPMI 1640 medium by supplementing with 10% fetal bovine serum (FBS), 5 x 10^-5 M β-mercaptoethanol, and 10 µg/mL gentamicin. Recombinant murine FL is also commercially available.

2. Lentiviral transduction

1. Plate iHSPCs at a density of 1 x 10⁵ cells/well in 12-well plates in 1 mL of complete medium containing 100 ng/mL FL, 1 µM β-estradiol, and 8 µg/mL polybrene.
   NOTE: The concentration of polybrene depends on the cell types and is usually in the range of 4-8 µg/mL.
2. Add shRNA-carrying lentivirus in each well at a multiplicity of infection (MOI) of 100.
   NOTE: The lentiviral vector is pLKO.1-Puro with a puromycin selection marker (Figure 1).The target sequences of shRNAs against LacZ, Tcf4, and Id2, respectively, are listed in the Table of Materials. The MOI is defined by the number of virions that are added per cell during infection.
   
3. Spin the plates at 1,100 x g for 90 min at 37 °C.
   NOTE: Spin infection is carried out using a swing bucket rotor.
4. Incubate the plate containing infected cells for overnight at 37 °C in an incubator.
   NOTE: If the cells are sensitive to polybrene, then refresh the cells with the complete medium without polybrene after the spin infection.
5. Refresh cells with complete medium containing 100 ng/mL FL and 1 µM β-estradiol 24 h after the infection.
6. Add 6 µg/mL of puromycin to the medium to select the infected cells after an additional 24 h.
   NOTE: Transduced lentiviral vector usually takes 48 h for expressing genes, including puromycin resistant gene. Ensure that the concentration of puromycin is optimized for each cell line.
7. Refresh the selection medium containing 100 ng/mL FL, 1 µM β-estradiol, and 6 µg/mL puromycin every 3 days and maintain the cells for at least one week to expand the stably transduced iHSPC cells.
   ​NOTE: Puromycin selection usually takes effect 48 h later, and the period of selection depends on cell types.

3. Measurement of knockdown efficiency by reverse transcription and real-time PCR (RT-PCR)

1. Extract total RNA from 1 x 10⁷ shLacZ, shTcf4, and shId2 stable knockdown iHSPC cells using commercial RNA extraction reagent and precipitate RNA from the aqueous layer with isopropanol, followed by washing the RNA precipitate with 75% ethanol.
2. Dissolve the RNA (~ 5 µg) with 5 µL of DEPC-treated H₂O and adjust the concentration to 1 µg/µL.
3. Take 1-3 µg of RNA, mix with DEPC-treated H₂O to a final volume of 17.4 µL, add 1 µL of 1 unit/µL RNase-free DNase I and incubate for 20 min at 37 °C.
   NOTE: This step is to digest the genomic DNA in the RNA samples.
4. Add 1 µL of 20 mM EDTA to the RNA samples, incubate at 65 °C for 10 min to inactivate DNase I, and put the RNA samples immediately at 4 °C.
5. Add 11.6 µL of the reaction mix containing 1 µL of oligo (dT) primers (45 µM), 6 µL of 5x 1^st strand buffer, 3 µL of dNTP (2 mM), 0.6 µL of RNase inhibitor (50 Unit/µL) and 1 µL of reverse transcriptase (200 Unit/µL) to the RNA samples and incubate at 40 °C for 1 h.
6. Stop the reaction by heating at 70 °C for 10 min and dilute the reaction mix with 30 µL of H₂O.
7. Take 2 µL of the diluted RT reaction mix as a DNA template and PCR amplify it using primers against Tcf4 or Id2 (see Table 1 for thermocycler conditions).
   ​NOTE: The primer sequences are included in the Table of Materials.

4. In vitro differentiation of the stable knockdown iHSPC cell lines

1. Maintain the stable single knockdown of LacZ (shLacZ), Tcf4 (shTcf4), or Id2 (shId2) in iHSPCs in complete medium containing 100 ng/mL of FL and 1 µM β-estradiol.
2. Collect shLacZ, shTcf4, and shId2 un-differentiated iHSPCs into a 15 mL tube and centrifuge for 5 min at 500 x g to pellet the cells.
3. Discard the supernatant and add 10 mL of PBS to wash the cells. Repeat this step twice.
4. Resuspend and seed the iHSPC cells at a density of 2 x 10⁵ cells/mL into a 12-well plate in 1 mL of complete medium containing 100 ng/mL FL only.
5. Add 1 mL of fresh complete medium containing 100 ng/mL FL three days later.
6. Analyze the differentiated cells (shLacZ, shTcf4, and shId2 iHSPCs) by flow cytometry two days later.

5. Flow cytometric analysis of the differentiated DCs

1. Collect the cells into 1.5 mL tubes by pipetting the cells up and down 2-3 times in the plate, and centrifuge for 5 min at 500 x g at room temperature (RT).
   NOTE: In vitro differentiated DCs will slightly attach to the plates. Gentle pipetting will help recover DCs from the plates.
2. Discard the supernatant and resuspend the cells in 50 µL of FACS buffer. Next, add 50 µL of anti-CD16/32 hybridoma supernatant and incubate for 5-10 min on ice.
   NOTE: The Fc blocking antibody prevents nonspecific binding of antibodies to Fc receptors expressing on some myeloid cells and B cells and is commercially available through various vendors.
3. Add fluorescent dye-conjugated antibodies (0.04 µg for each antibody) directly to the cells and incubate for 15 min on ice in the dark. The antibodies used are APC/cy7 anti-mouse CD11c, FITC anti-mouse CD11b, and PE anti-mouse B220.
   NOTE: pDCs are defined as CD11c⁺CD11b^–B220⁺, and cDCs are defined as CD11c⁺CD11b⁺B220^–.
4. Wash cells with 1 mL of FACS buffer and centrifuge for 5 min at 500 x g at RT.
5. Resuspend the cells in 100 µL of FACS buffer and analyze by flow cytometry.