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

Begin with an immortalized hematopoietic stem and progenitor cell, iHSPC, culture in media containing specific steroid hormone and hematopoietic cytokine, regulating cell proliferation in an undifferentiated state. Add a suitable cationic polymer and lentiviral vectors.

Recombinant viruses carry coding sequences for short hairpin RNAs, shRNAs, directed toward a specific host gene and antibiotic resistance marker gene.

Centrifuge to sediment cells and viruses. During incubation, cationic polymers neutralize the cellular membrane charges, enhancing viral binding. Following membrane fusion, viral genetic material and enzymes release into the cellular cytoplasm. The viral genome is reverse transcribed, moves into the nucleus, and integrates into the host genome.

Under a specific promoter's influence in the construct, the shRNA gene construct comprising a target mRNA matching sequence, loop segment, and another sequence complementary to the first, is transcribed forming shRNA with loop segment of unpaired nucleotides. Following transcription, shRNA is exported to the cytoplasm.

A specific ribonuclease cleaves the loop segment, forming double-stranded RNA, siRNA. siRNA is incorporated into the RNA-induced silencing complex, RISC, and siRNA strands separate. The activated complex with the retained strand complementary to the target mRNA recognizes and binds to the target mRNA in a sequence-specific manner, negatively regulating expression and mediating target gene suppression.

Incubate the cells with specific antibiotics. The stably transduced cells express the antibiotic resistance gene allowing their selection. Refresh media and incubate, allowing proliferation of stably transduced cells.

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.