JoVE Encyclopedia of Experiments
Microbiology
0 views • 3:33 min • August 31st, 2026
Take a human cell culture.
Add lentiviruses encoding a conditional host mRNA–targeting shRNA under a tetracycline-responsive promoter fused to an operator.
The lentivirus also carries genes for a tetracycline repressor and an antibiotic-resistance protein, placed under a constitutive promoter.
Incubate to allow delivery of viral RNA into host cells.
The RNA converts to cDNA and integrates into the host genome.
Wash, add media and incubate to allow expression of the tetracycline repressor and the antibiotic-resistance protein, generating transduced cells. The tetracycline repressor binds to the operator and suppresses shRNA expression.
Wash, add antibiotic-supplemented media and incubate.
Transduced cells express the antibiotic resistance protein, enabling their selective survival in the presence of the antibiotic.
Add doxycycline, a tetracycline analog, to the culture.
Doxycycline binds to the tetracycline repressor, preventing its binding to the operator and enabling shRNA expression.
The shRNA is processed into siRNA, which loads into RISC and degrades the target mRNA.
This generates doxycycline-dependent conditional knockdown cells.
To generate stable cell lines, start by seeding HCT-116 co
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