shRNA gene silencing Human - SiHa MCM4

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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5 years ago

5 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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Found 1 matching solution for this experiment

MCM4 shRNA (h) Lentiviral Particles

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	Cervical cancer cell lines, viz. SiHa, used for this study were kind gift from Dr. VVS Murty, Columbia University, New York, USA, and grown in recommended media supplemented with 10 % fetal bovine serum and 1 % antibiotic cocktail. These cell lines had been extensively characterized [13, 14]. To confirm the HPV status of these cell lines, we performed HPV genotyping using typespecific primers (Table 1). MCM4 short hairpin RNA (shRNA) lentiviral particles werepurchased from Santa Cruz Biotechnology. This is a pool of transduction ready viral particles containing three targetspecific constructs that encode 19–25 nucleotide (plus hairpin) shRNA designed to knock down MCM4 gene expression. Lentiviral particles containing shRNA construct encoding a scrambled sequence (Santa Cruz Biotechnology,USA) were used as a negative control that will not lead to the specific degradation of any known cellular mRNA. Cells were transfected with shMCM4 and scrambled lentiviral particles in medium supplemented with 5 μg/ml polybrene, according to supplier’s protocol. copGFP lentiviral particles (Santa Cruz Biotechnology) were used as a positive control to determine the optimal delivery condition. The amount of lentiviral particles used for the cells was optimized after trying several concentrations. Stable clones expressing shRNA were selected by puromycin dihydrochloride selection. shRNA lentiviral particles are compatible to maintain MCM4 downregulation for the subsequent generations (data not shown). Same generations of cells were used for a subset of experiments.

Protocol tips
Cervical cancer cell lines, viz. SiHa, used for this study were kind gift from Dr. VVS Murty, Columbia University, New York, USA, and grown in recommended media supplemented with 10 % fetal bovine serum and 1 % antibiotic cocktail. These cell lines had been extensively characterized [13, 14]. To confirm the HPV status of these cell lines, we performed HPV genotyping using typespecific primers (Table 1). MCM4 short hairpin RNA (shRNA) lentiviral particles werepurchased from Santa Cruz Biotechnology. This is a pool of transduction ready viral particles containing three targetspecific constructs that encode 19–25 nucleotide (plus hairpin) shRNA designed to knock down MCM4 gene expression. Lentiviral particles containing shRNA construct encoding a scrambled sequence (Santa Cruz Biotechnology,USA) were used as a negative control that will not lead to the specific degradation of any known cellular mRNA. Cells were transfected with shMCM4 and scrambled lentiviral particles in medium supplemented with 5 μg/ml polybrene, according to supplier’s protocol. copGFP lentiviral particles (Santa Cruz Biotechnology) were used as a positive control to determine the optimal delivery condition. The amount of lentiviral particles used for the cells was optimized after trying several concentrations. Stable clones expressing shRNA were selected by puromycin dihydrochloride selection. shRNA lentiviral particles are compatible to maintain MCM4 downregulation for the subsequent generations (data not shown). Same generations of cells were used for a subset of experiments.

Protocol tips

Cervical cancer cell lines, viz. SiHa, used for this study were kind gift from Dr. VVS Murty, Columbia University, New York, USA, and grown in recommended media supplemented with 10 % fetal bovine serum and 1 % antibiotic cocktail. These cell lines had been extensively characterized [13, 14]. To confirm the HPV status of these cell lines, we performed HPV genotyping using typespecific primers (Table 1). MCM4 short hairpin RNA (shRNA) lentiviral particles werepurchased from Santa Cruz Biotechnology. This is a pool of transduction ready viral particles containing three targetspecific constructs that encode 19–25 nucleotide (plus hairpin) shRNA designed to knock down MCM4 gene expression. Lentiviral particles containing shRNA construct encoding a scrambled sequence (Santa Cruz Biotechnology,USA) were used as a negative control that will not lead to the specific degradation of any known cellular mRNA. Cells were transfected with shMCM4 and scrambled lentiviral particles in medium supplemented with 5 μg/ml polybrene, according to supplier’s protocol. copGFP lentiviral particles (Santa Cruz Biotechnology) were used as a positive control to determine the optimal delivery condition. The amount of lentiviral particles used for the cells was optimized after trying several concentrations. Stable clones expressing shRNA were selected by puromycin dihydrochloride selection. shRNA lentiviral particles are compatible to maintain MCM4 downregulation for the subsequent generations (data not shown). Same generations of cells were used for a subset of experiments.

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