Short Hairpin RNA-Mediated Gene Knockdown in iHSPCs In Vitro: A Lentivirus-Based shRNA Expression System Delivery into iHSPCs for Knockdown of Specific Gene Expression

To begin, maintain the immortalized hematopoietic stem and progenitor cells or iHSPCs in complete RPMI 1640 medium supplemented with 100 nanograms per milliliter of the Flt3 ligand, and one micromolar beta-estradiol. For lentiviral transduction, plate the iHSPCs in 12-well plates at a density of 1 x 10⁵ cells per well and one milliliter of complete medium containing the Flt3 ligand, beta-estradiol, and polybrene.

Then, add the lentivirus carrying the short hairpin RNA in each well at a multiplicity of infection of 100. Next, spin the plate at 1,100 x g and 37 degrees Celsius for 90 minutes. Then, incubate the infected cells overnight at 37 degrees Celsius. The following day, remove the polybrene by collecting the cells into 15-milliliter tubes and centrifugation, following replacing the media with fresh complete medium containing the Flt3 ligand and beta-estradiol.

After an additional 24 hours, add six micrograms per milliliter of puromycin to the medium to select the infected cells. Replace the selection medium every three days and maintain the cells for at least one week to expand the stably transduced iHSPCs.