Method and Apparatus for Extracting Molecular Species from Intact Cells

Reference Number(s)
     Laboratory:  214
     DOE reference no.(s): 105,082

Abstract

Methods and apparatus are described that enable the extraction of molecular species from intact, viable cells. Vertically aligned arrays of needles, spikes, pipes and partial pipes are described that feature radial dimensions small enough to penetrate within the smallest of cells, but are long enough to provide a significant penetration depth into cells and sub-cellular regions. Surface modification and other functionalization strategies are described which allow the penetrant structure to capture intracellular molecular species and effectively remove them from the viable, penetrated cell.

Application(s) of the Technology

Functional Genomics: Method to extract specific proteins from cells for analysis of their functionality. Protein is extracted from native context, not context of cell lysate. Provider cell can be located following extraction and proliferated to provide more protein.

Pharmaceutical: Method to sample cells for production of specific protein. Combinatorial methods could be used to produce different proteins or different protein combinations in cells. The array of strains could be screened with this technique and the cell that produces a desired protein could be isolated and cloned.

Gene Array Analysis: Method to sample mRNA from cell without requiring lysis of cells. This could provide multiple read points for mRNA analysis of cells. Cells remain viable, so cells could be sampled and dynamic mRNA analysis could be performed as opposed to current methods which only provide a single point in time due to requirement to lyse cells. Also, specific antisense could be tethered to capture specific mRNAs for dynamic (albeit not real time) visualization of gene expression.

Cancer Screening: Tissue could be sampled possibly in-vivo for specific proteins that are present or absent due to tumorigenesis. Spatial sampling could identify for the surgeon the extent of the pathology such that all tumorigenic tissue is removed. Resampling can be performed without excessive damage of tissue.

Industrial: Efficient harvesting of commercially produced enzymes/proteins/co-factors. Rather than growing up and lysing cells to harvest, could conceivably harvest repeatably from same culture. Purification may be less costly due to specific extraction from intracellar domain, rather than from oxidatively stressed lysate.

Molecular Toxicology and Toxicological screening: Extraction of protein adducts generated due to contaminant exposure. These sampled cells could be proliferated to see if exposure and generation of adducts results in pathology, and this could be correlated directly to quantity of adduct found in those specific cells. Alternative is lysing sample of cells and observing like treated cells, which can potentially generate error in interpretation.

Main Advantages of Invention

This process provides the potential for massively parallel embodiment, extraction from many cells simultaneously. It provides the ability to electrically address and therefore provide additional functionality such as capture stringency, capture and release, tether retraction and casting. It uses a sequestered environment of pipe to reduce shear during insertion and extraction, and the uUse of a hybrid penetrant/tether to provide penetration but still allow ‘fishing’ within intracellular domain to provide higher capture efficiency. Through the use of cellular targeting sites on structure to localize fishing line to specific regions of the cell (i.e. nucleus for example), does not kill sampled cell, thus allowing resampling and proliferation of cells following extraction.

In contrast to traditional lysis and affinity capture, provides affinity capture within an intact viable cell. This allows the protein to be captured in its native context (not after suffering alterations due to lysis, and non-intracellular residence), it enables the probed cell to be resampled and potentially propagated further in response to the extraction result.

Status/Availability

Available both Exclusively and Non Exclusively

Patents & Applications

Posting Date:  Monday, Feb 6 2006

Contact

Mark E Reeves
Oak Ridge National Laboratory
P.O. Box 2008
Building /4500N, Room 145
Mailstop 6196
Oak Ridge, TN 37831
(865) 576-2577
reevesme@ornl.gov