Targeted gene silencing in human embryonic stem cells using cell-penetrating peptide PepFect 14 (2019)
Sequence: stearyl-AGYLLGKLLOOLAAAALOOLL-NH2
| Experiment Id | EXP001462 |
|---|---|
| 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 | no |
| 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 | 30 nM siRNA-Alexa Fluor 568 (typical uptake experiment) |
| 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 | Uptake/internalization by flow cytometry (4–32 h) and confocal microscopy (24 h) |
| Output Value | |
| Output Units | |
| Output Notes | Flow cytometry: ~91% AF568+ cells at 4 h; ~91.9% at 8 h; ~89.4% at 24 h; ~82% at 32 h (8 h after medium exchange). Confocal microscopy showed intracellular AF568 signal after 24 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 |