shRNA gene silencing Human - THP-1 TLR10

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi has been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining the efficacy of transduction and shRNA on its target site.

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4 years ago

4 years ago by Eufrosina Sagese 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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TLR10 shRNA (h) Lentiviral Particles

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream 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.

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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

TLR10 shRNA (h) Lentiviral Particles

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream 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.

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.

Protocol tips

Manufacturer protocol Publication protocol 1 Relevant paper

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