shRNA gene silencing Human - TF‐1 AChE

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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Found 1 matching solution for this experiment

AChE shRNA Plasmids (h)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	TF‐1 cells (CRL‐2003; ATCC; Manassas, VA; RRID:CVCL_0559), an erythroleukemic cell line from human blood, were maintained at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mM sodium pyruvate, 4.5 g/L glucose and 2 ng/mL GM‐CSF (PHC2015). Prior to induction of differentiation, cells were kept in medium without GM‐CSF overnight, followed by the addition of EPO (1 U/mL; PHC2054) on the next day. The TF‐1 cell line is not listed in the database of commonly misidentified cell lines (International Cell Line Authentication Committee), and not authenticated by the authors. All culture reagents were from Life Technologies (Life Technologies, Carlsbad, CA, USA). The renewal of medium and EPO (1 U/mL) were done on alternative days, and the cell density was maintained at 4 × 105 cells/mL. Cultured TF‐1 cells were transfected with cDNA constructs with Trans IT X2® Dynamic Delivery System (Mirus BIO, Madison, WI, USA) for AChE shRNA and (Santa Cruz, Dallas, TX, USA). Transfection was performed 1 day after cells were plated. For every 5 × 106 cells in 1 mL medium, DNA: Trans IT X2® were mixed in the ratio of 1 : 2 (3 μg/6 μL) in antibiotic free medium and added to the cells within 20 min.

Protocol tips
TF‐1 cells (CRL‐2003; ATCC; Manassas, VA; RRID:CVCL_0559), an erythroleukemic cell line from human blood, were maintained at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mM sodium pyruvate, 4.5 g/L glucose and 2 ng/mL GM‐CSF (PHC2015). Prior to induction of differentiation, cells were kept in medium without GM‐CSF overnight, followed by the addition of EPO (1 U/mL; PHC2054) on the next day. The TF‐1 cell line is not listed in the database of commonly misidentified cell lines (International Cell Line Authentication Committee), and not authenticated by the authors. All culture reagents were from Life Technologies (Life Technologies, Carlsbad, CA, USA). The renewal of medium and EPO (1 U/mL) were done on alternative days, and the cell density was maintained at 4 × 105 cells/mL. Cultured TF‐1 cells were transfected with cDNA constructs with Trans IT X2® Dynamic Delivery System (Mirus BIO, Madison, WI, USA) for AChE shRNA and (Santa Cruz, Dallas, TX, USA). Transfection was performed 1 day after cells were plated. For every 5 × 106 cells in 1 mL medium, DNA: Trans IT X2® were mixed in the ratio of 1 : 2 (3 μg/6 μL) in antibiotic free medium and added to the cells within 20 min.

Protocol tips

TF‐1 cells (CRL‐2003; ATCC; Manassas, VA; RRID:CVCL_0559), an erythroleukemic cell line from human blood, were maintained at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mM sodium pyruvate, 4.5 g/L glucose and 2 ng/mL GM‐CSF (PHC2015). Prior to induction of differentiation, cells were kept in medium without GM‐CSF overnight, followed by the addition of EPO (1 U/mL; PHC2054) on the next day. The TF‐1 cell line is not listed in the database of commonly misidentified cell lines (International Cell Line Authentication Committee), and not authenticated by the authors. All culture reagents were from Life Technologies (Life Technologies, Carlsbad, CA, USA). The renewal of medium and EPO (1 U/mL) were done on alternative days, and the cell density was maintained at 4 × 105 cells/mL. Cultured TF‐1 cells were transfected with cDNA constructs with Trans IT X2® Dynamic Delivery System (Mirus BIO, Madison, WI, USA) for AChE shRNA and (Santa Cruz, Dallas, TX, USA). Transfection was performed 1 day after cells were plated. For every 5 × 106 cells in 1 mL medium, DNA: Trans IT X2® were mixed in the ratio of 1 : 2 (3 μg/6 μL) in antibiotic free medium and added to the cells within 20 min.

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