EXP001463

Paper

Targeted gene silencing in human embryonic stem cells using cell-penetrating peptide PepFect 14 (2019)

Peptide

Sequence: stearyl-AGYLLGKLLOOLAAAALOOLL-NH2

RNA

All experiment fields

Experiment Id	EXP001463
Paper	Targeted gene silencing in human embryonic stem cells using cell-penetrating peptide PepFect 14
Peptide	PepFect 14 (PF14)
Delivery Success Class	no
In Vivo Flag	no
Uptake Confirmed	yes
Label Confidence	high
In Vitro Functional Effect	yes
Endosomal Escape Evidence
Peptide Concentration	Set by CR 2:1 and MR 16:1 (peptide:siRNA); PF14 stock 100 µM used. (Exact µM peptide depends on siRNA concentration.)
Rna Concentration	10–30 nM siOCT4 (optimized 20 nM for best balance of silencing + viability)
Mixing Ratio	Complexes formed in 200 µL (1/10 final transfection volume) for 1 h at room temperature; adjusted to charge ratio (CR/N:P) 2:1 and molar ratio (MR) 16:1 (peptide:siRNA).
Formulation Format	PF14/siRNA nanocomplexes (noncovalent electrostatic nanoparticles)
Formulation Components	PepFect 14 (PF14) + siRNA (targeted or control) in mTeSR1 medium; Transfection performed immediately after EDTA passaging and plating hES cells as small clumps in mTeSR1 + ROCK inhibitor (Y-27632, 10 µM). Complexes kept in medium 24 h, then medium exchanged.
Size Nm
Zeta Mv
Model Scope	in_vitro
Model Type	in vitro
Cell Lines Or Primary Cells	Human embryonic stem cells: H9 (WA09)
Animal Model
Administration Route
Output Type	In vitro functional effect: OCT4 knockdown (flow cytometry protein; RT-qPCR mRNA) at 48–72 h
Output Value
Output Units
Output Notes	Protein: ~70% of cells became OCT4-negative (≈25% OCT4+ vs ≈90% in controls) at 48 h and 72 h. mRNA: OCT4 reduced up to ~90% in bulk population (20–30 nM) by RT-qPCR; dosage effect observed (e.g., 10 nM less sustained at 72 h).
Toxicity Notes	Cell viability decreased at higher siRNA/PF14 doses (30 nM), while 10–20 nM maintained viability close to untreated controls (cell count at 72 h).
Curation Notes