Generating Conditional Knockdown Cells Using a Tetracycline-Responsive Lentiviral shRNA Expression System

Abstract

Source: Kubala, M. H. & DeClerck, Y. A. Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment. J. Vis. Exp. (2017)

This video demonstrates the generation of conditional knockdown cancer cell lines using a Tet-inducible lentiviral shRNA system. It outlines the steps for viral transduction, antibiotic selection, and doxycycline-induced gene silencing.

Protocol

1. Generation of stably transduced cell lines
   1. Seed HCT116 colon cancer cells and MDA-MB-231 breast cancer cells at 50,000 cells/well in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Pen/Strep) in a 12-well plate. Incubate at 37 °C at 5% CO₂ until cells are 60 - 70% confluent.
   2. Wash cells with phosphate-buffered saline (PBS) and add 1 mL of virus-containing medium to cancer cells and incubate overnight at 37 °C at 5% CO₂. As a control, add 1 mL of fresh DMEM medium supplemented with 10% FBS and 1% Pen/Strep to 2 wells with cancer cells.
   3. Carefully remove virus-containing medium by aspiration. Wash the cells with PBS and change the medium to DMEM with 10% (v/v) tetracycline-free (Tet-free) FBS and 1% Pen/Strep.
   4. After 72 h, wash cells with PBS and change the medium to DMEM 10% Tet-free FBS 1% Pen/Strep, 1 µg/mL puromycin for selection of virus-transduced cells.
      1. Depending on the cell line used, adjust the puromycin concentration for optimal selection by testing the range of puromycin concentrations (0.1, 0.5, 1, 2, and 5 µg/mL) in non-transduced cells and choosing the lowest concentration where no control cells survive after 3 days of culture.
   5. Select virus-transduced cells by culturing cells in the presence of puromycin for 3 - 14 days. As controls, prepare two additional wells with non-transduced cells, one with medium containing puromycin and one without puromycin. Observe partial killing of the cells by puromycin in the well with virus-transduced cells compared to the control wells.
      NOTE: Non-transduced cells will not grow in the presence of puromycin.
   6. Verify the efficiency of the conditional knockdown (KD) in the puromycin-resistant HCT116 colon cancer and MDA-MB-231 breast cancer cells.
      NOTE: The effective concentration of doxycycline will vary with the cell line used and must be titrated (typically from 100 ng/mL to 2 µg/mL).
      1. Determine the doxycycline concentration that is not toxic for the cells but effective in downregulating the expression of the protein of interest. In this protocol, 1 µg/mL was successfully used in vitro.
      2. Culture the cells for 72 h in the presence and absence of 0.1, 1, and 2 µg/mL doxycycline. Verify the level of expression of the downregulated protein (here, plasminogen activator inhibitor 1 (PAI-1)) in the cell lysate by Western blot analysis. Compare HCT116 and MDA-MB-231 cells conditionally expressing short hairpin RNA (shRNA) to scrambled control cells.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tet-pLKO-puro lentiviral vector	Addgene	21915
FBS (Tetracycline-free)	Omega Scientific	FB-15
psPAX vector	Addgene	12260
pMD2.G vector	Addgene	12259
HEK293 cells	ATCC	CRL-1573
PBS	Corning	21-031-CV
Dulbecco’s Modified Eagle’s Medium (DMEM)	Corning	10-013-CV
Phosphate-buffered saline (PBS)	Corning	21-031-CV
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Sigma-Aldrich	H3375
Puromycin	Sigma-Aldrich	P9620
Doxycycline hyclate	Sigma-Aldrich	D9891-25g
Roswell Park Memorial Institute (RPMI)	Corning	10-040-CV
Penicillin-Streptomycin	Gibco	15140122