JoVE Encyclopedia of Experiments
Microbiology
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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 colon cancer cells and MDA-MB-231 breast cancer cells into a 12-well plate and 50,000 cells per well.
Incubate at 37 degrees Celsius and 5%carbon dioxide. When the cells are 60-70%confluent, wash the cells with PBS and add one milliliter of virus-containing medium to each well. The next day, carefully remove the virus-containing medium by aspiration.
Then after washing the cells with PBS, add medium containing tetracycline-free FBS. After 72 hours, wash the cells as before and add medium supplemented with one microgram per milliliter puromycin for the selection of virus-transduced cells. Select virus-transduced cells by culturing cells in the presence of puromycin for three to 14 days.
Observe partial killing of the cells by puromycin in the wells with virus-transduced cells. Non-transduced cells will not grow in the presence of puromycin.
Verify the efficiency of the conditional knockdown in the puromycin-resistant HCT-116 colon cancer and MDA-MB-231 breast cancer cells by adding medium with different concentrations of doxycycline and culturing the cells for 72 hours.