Clarke MS, Vanderburg CR, Carpenter J, Howerte C, Abbasi T, Feeback DL.
Mol Biol Cell. 1999 Nov; 10 Suppl: 270a.
Universities Space Research Association, Houston, TX 77058, USA.
We have previously described an impact-mediated loading (IML) technique capable of loading macromolecules into living cells via mechanically induced transient plasma membrane disruptions known as membrane wounds. We here report the development of an IML device, known as the g-Loader, capable of performing this procedure under hypergravity conditions (Range 2-200xg). Utilizing fluorescently labeled dextrans (FDx), greater than 95% of the monolayer can be loaded with FDx having a Mr of 2 million daltons or below. Hypergravity enhances the amount of FDx loaded into individual cells due to a process where increased g-force promotes movement of macromolecules through the open membrane wound. Utilizing the g-Loader, a range of macromolecules, including FDx (10-2000 kD), IgG, calcium indicators and oligonucleotides have been efficiently loaded into various cell types, including primary human myoblasts, primary chick corneal fibroblasts, bovine endothelial cells, C2C12 myoblasts, NIH 3T3 fibroblasts and NGF-differentiated PC-12 cells. Additionally, the g-Loader can be used to transfect all of the above cell types with plasmid DNA. Depending on cell type, transient transfection efficiency is between 15% and 30%. This technique for macro-molecular loading is based on a mechanistic approach whereby each experimental parameter, including wound frequency, wound severity, macromolecule concentration and g-force can be quickly and easily optimized for a particular cell type.
Publication Types:
Keywords:
- Animals
- Cattle
- Dextrans
- Fibroblasts
- Humans
- Hypergravity
- Macromolecular Substances
- Plasmids
- Transfection
- genetics
- instrumentation
- NASA Discipline Cell Biology
- Non-NASA Center
Other ID:
UI: 102194976
From Meeting Abstracts