TLR10 shRNA (h) Lentiviral Particles

shRNA gene silencing Human - THP-1 TLR10

Experiment
shRNA gene silencing Human - THP-1 TLR10
Product
TLR10 shRNA (h) Lentiviral Particles from Santa Cruz Biotechnology
Manufacturer
Santa Cruz Biotechnology

Protocol tips

Protocol tips
The human monocytic cell line THP-1 was obtained from ATCC (Manassas, VA, USA). THP-1 cells were grown in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% Antibiotic-Antimycotic, 10 mM HEPES buffer, and β-mercaptoethanol at 37 °C in a 5% CO2 humidified incubator.TLR10 knockdown THP-1 cell lines were established using stable expression of short hairpin RNAs (shRNAs) that target TLR10 mRNA. THP-1 cells were transduced with TLR10 shRNA lentiviral particles (sc-40272-V), control shRNA lentiviral particles (sc-108080), or copGFP control lentiviral particles (sc-108084) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).Transductions of THP-1 cells were carried out in the presence of 8 µg/mL of polybrene (sc-134220, Santa Cruz (Dallas, TX, USA)). After mixing with polybrene, the viral stocks were added to the cells (1 × 104 cells/well in 96 well plates) at multiplicity of infection (MOI) of 10. After 24 h of transduction, the cells were collected and then fresh media lacking polybrene were added to them. The transduced cells were allowed to proliferate until a sufficient cell number was reached for puromycin selection, which was performed in order to select stable clones expressing the shRNA. The cell culture medium was replaced with fresh medium plus 1 µg/mL of puromycin every 2 to 3 days until resistant clones appeared. After 3–4 weeks, the cells were collected and examined for GFP and TLR10 expression of the cells using FACS analysis. The selected clones were maintained in fresh puromycin-containing medium for an additional month, analyzed, and used for further experiments.

Publication protocol

The human monocytic cell line THP-1 was obtained from ATCC (Manassas, VA, USA). THP-1 cells were grown in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 1% Antibiotic-Antimycotic, 10 mM HEPES buffer, and β-mercaptoethanol at 37 °C in a 5% CO2 humidified incubator.TLR10 knockdown THP-1 cell lines were established using stable expression of short hairpin RNAs (shRNAs) that target TLR10 mRNA. THP-1 cells were transduced with TLR10 shRNA lentiviral particles (sc-40272-V), control shRNA lentiviral particles (sc-108080), or copGFP control lentiviral particles (sc-108084) purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).Transductions of THP-1 cells were carried out in the presence of 8 µg/mL of polybrene (sc-134220, Santa Cruz (Dallas, TX, USA)). After mixing with polybrene, the viral stocks were added to the cells (1 × 104 cells/well in 96 well plates) at multiplicity of infection (MOI) of 10. After 24 h of transduction, the cells were collected and then fresh media lacking polybrene were added to them. The transduced cells were allowed to proliferate until a sufficient cell number was reached for puromycin selection, which was performed in order to select stable clones expressing the shRNA. The cell culture medium was replaced with fresh medium plus 1 µg/mL of puromycin every 2 to 3 days until resistant clones appeared. After 3–4 weeks, the cells were collected and examined for GFP and TLR10 expression of the cells using FACS analysis. The selected clones were maintained in fresh puromycin-containing medium for an additional month, analyzed, and used for further experiments.

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Discussion

Discussion

5 years ago

Author: Italy

Is a knockdown using shRNA permanent?

Is a knockdown using shRNA permanent and if not is there a known duration?

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Papers

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Manufacturer protocol

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