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Self-delivering AUMsilence sdASO

Targeted mRNA knockdown, from cell culture to in vivo studies

No lipid-based transfection reagent, electroporation, or viral vector required.

In an animal the same AUMsilence sdASO is given in saline or PBS, by the routes below, with no delivery vehicle, no lipid nanoparticle and no viral vector. Testing a dose range of 3-30 mg/kg in mice is recommended.

T cells

CD4+ (naive, activated, memory, exhausted), CD8+ (naive, effector, memory, exhausted), Tregs, Th1, Th2, Th17, Tfh, gd T cells, NKT, MAIT, CAR-T cells, TILs

B cells and NK cells

Naive B cells, Memory B cells, Plasma cells, GC B cells, Primary NK cells (CD56+CD3-), CAR-NK cells

Macrophages, DCs and granulocytes

Monocytes, M1/M2 macrophages, BMDMs, Peritoneal, Alveolar, Kupffer cells, Microglia, TAMs, Myeloid DCs, Plasmacytoid DCs, Neutrophils, Eosinophils, Basophils, Mast cells

Hematopoietic progenitors

Long-term HSCs, Short-term HSCs, CD34+ HSPCs, MPPs, CLPs, CMPs, GMPs, MEPs, AGM embryonic HSCs

Neurons

Cortical, Hippocampal, Motor, Sensory, DRG, Dopaminergic, GABAergic, Glutamatergic, Purkinje, Retinal ganglion, Enteric, iPSC-derived neurons

Glial and neural stem cells

Astrocytes, Oligodendrocytes, Microglia, Schwann cells, hiPSC-derived NSCs, Adult neural progenitors, Hippocampal slices, Brain sections, Spinal cord explants

Cardiovascular

Primary and iPSC cardiomyocytes, HASMCs, HCASMCs, Pulmonary artery SMCs, Endothelial cells (HUVECs, HAECs), Cardiac fibroblasts, Pericytes

Musculoskeletal

Myoblasts, Myotubes, Satellite cells, Osteoblasts, Osteoclasts, Osteocytes, Chondrocytes, Synoviocytes, Tenocytes, MSCs

Stem and progenitor cells

Human ESCs, Mouse ESCs, iPSCs, iPSC-derived neurons, cardiomyocytes, hepatocytes, NSCs, HSCs, CD34+ HSPCs, MSCs, Cardiac and Endothelial progenitors, Intestinal stem cells (Lgr5+)

Liver and gastrointestinal

Primary hepatocytes, Hepatic stellate cells, Cholangiocytes, Intestinal epithelial, Colonocytes, Paneth cells, Goblet cells, Pancreatic beta, ductal, and acinar cells

Lung, airway and kidney

Tracheobronchial epithelial, Alveolar epithelial type I and II, Bronchial epithelial, Airway smooth muscle, Lung fibroblasts, Podocytes, Mesangial cells, Tubular epithelial cells

Skin, eye, reproductive and other

Keratinocytes, Melanocytes, Fibroblasts, CAFs, RPE cells, Corneal epithelial, Trophoblasts, HEK293T, NIH3T3, THP-1, Jurkat, Neuro-2a

  • No transfection reagents
  • No viral vectors
  • No electroporation
  • Suspension and adherent cells

Protocol

In vitro workflow

Optimize cell density, AUMsilence sdASO concentration, exposure duration, and assay time point for each model.

  1. Step 1, prepare cells and the oligonucleotide: a clear 24-well plate of pink medium, a microtube of pale blue solution with its lid open and a pipette tip reaching into its mouth, and three short beaded violet strands drawn in the air above the tube, the slide's motif for the oligonucleotide.

    Step 1

    Prepare cells and AUMsilence sdASO

    Plate cells at the optimized density. Reconstitute AUMsilence sdASO in sterile, nuclease-free water or a validated compatible buffer.

  2. Step 2, add to the culture medium: a single-channel micropipette with a violet plunger held over a 24-well plate of pink medium, its tip in one well.

    Step 2

    Add to culture medium

    Add AUMsilence sdASO directly to the culture medium at the study concentration.

  3. Step 3, incubate: a benchtop carbon dioxide incubator with its outer door and its inner glass door open, a 24-well plate of pink medium on the upper of two wire shelves, and a display band above the chamber with a violet and teal sweep at its left and a ring symbol on a dark panel at its right.

    Step 3

    Incubate

    Use a model-specific exposure period under standard culture conditions.

  4. Quantify knockdown: a schematic of three readouts, RT-qPCR, an immunoblot and flow cytometry, with no valuesThree small panels. An amplification curve pair for RT-qPCR, the control curve rising before the treated curve, with the axes named cycle and fluorescence and no tick values. An immunoblot with a control lane and a treated lane, a target band lighter in the treated lane and a loading control band alike in both, with no size marks. Two overlapping flow cytometry histograms, the treated one a little to the left, with the axes named fluorescence and count and no values. A schematic of the three readouts, not data.RT-qPCRcyclefluorescenceIMMUNOBLOTtargetloading controlcontroltreatedFLOW CYTOMETRYfluorescencecountcontroltreated

    Step 4

    Quantify knockdown

    Quantify target mRNA and, where appropriate, protein abundance or a functional phenotype. Include untreated and non-targeting controls.

    Schematic of the three readouts, not data. mRNA by RT-qPCR, protein by immunoblot, protein or phenotype by flow cytometry.