Our laboratories provide students and collaborators access to a variety of biotechnologies and experience, including studies on gene expression, bionanotechnology, BioMEMS, flow cytometry/rare event cell sorting, LEAP, and data mining. Dr. Leary has had over 30 years of multidisciplinary research experience with funding sources from NIH, NSF, DOE, and NASA.
I. High-Throughput Technologies
High Resolution Cell Sorting System (HiReCS)
Sorting of thioaptamer combinatorial library beads with bound P50. The "Brightest" bead subpopulations was the region where the sort gates were set. The goal was to sort 100 single beads for further DNA sequencing. (Leary, James F., Reece, Lisa M., Yang, Xianbin, Gorenstien, David. "High-Throughput Flow Cytometry Screening of Combinatorial Chemistry Bead Libraries for Proteomics and Drug Discovery". Advanced Biomedical and Clinical Diagnostic Systgems III, edited by Tuan Vo-Dinh, Warren S. Grundfest, David A. Benaron, Gerald E. Cohn, Proc. of SPIE Vol. 5692, 216-233)
Laser-Enabled Analysis and Processing (LEAP™)
Selective optoinjection of adherent cells (HeLa) and suspension cells (KG-1a). In Panels A-C brightfield (left) and darkfield (right) images of the same views were compared. A:Indirect optoinjection. Fluorescent dextrans were added to the medium prior to LEAP ablation of confluent cell monlayers, Panels 1-6. Untargeted cells surrounding the ablated areas were found to be optoinjected. Different geometrical shapes were targeted using the optimized protocol for controlled cell optoinjection to test injection efficacy. B:Optoinjection of confluent HeLa cells. Panels 1 and 2. A square pattern was shot using high laser power. The target area was ablated and indirect optoinjection occurred in the range of 8-12 cell diameters from the target area (region 1). Panels 3 and 4. A square pattern shot using low laser power. The target area was optoinjected with very few cells ablated (region 1). Panels 5 and 6. High magnification (40x) images of the optoinjected area. Most of the dextrans are observed in the cytoplasm, while the nuclei were visibly darker. C:Confocal images of optoinjected suspension cells. Panels 1 and 2. All cells within the targeted square area (dashed square) were optoinjected. Panels 3&4, Higher magnification (63x) images of the optoinjected area showed a visual difference between the dextran uptake of individual cells. D&E:Optical slices of the optoinjected area. Serial images of the same view at consecutive planes confirm that the dextrans were localized within the cells, after LEAP. (Peter Szaniszlo, William A. Rose, Nan Wang, Lisa M. Reece, Tamara V. Tsulaia, Elie G. Hanania, Cornelis J. Elferink, James F. Leary. " Scanning Cytometry with a LEAP: Laser-Enabled Analysis and Processing of Live Cells In Situ", Cytometry Special Issue, accepted Dec. 2005.)
Engineered multilayered nanoparticles targeted to radiation-damaged cells can initiate repair of damaged DNA using DNA repair genes manufactured inside individual living cells under the control of molecular biosensor switches. (Prow, T.W. Thesis. University of Texas Medical Branch, 2004)
III. Cancer Diagnostics and Therapeutics
Flow cytometric results from a defined cell mixture of 10-5 frequency MCF-7 cells in a major population of humen CEM/C7 T cells. Cells were labeled with a phycoerythrin (PE)-conjugated anti-CD45 antibody and a fluorescein isothiocyanate (FITC)-conjugated anti-cytokeratin antibody. A small subpopulation of rare MCF-7 cells was detected in region R2 in an aliquot of the sampe. Cells in this region were then sorted at the single-cell level for subsequent PCR analysis, TA cloning, and DNA sequencing. The four tumor cells, shown in this aliquot of cell sample, have been highlighted as dark enlarged circles in the flow cytometric distribution for easier viewing. The right-handed panel shows ECL detection of PCR-amplified sequences from sorted, rare, single tumor cells as shown in the left-hand panel. The top subpanel shows nested PCR product on 2% agarose gel stained with ethidium bromide. The lower subpanel shows the result of the ECL Southern blotting with HLA DQa type 4 probe. The result indicated that the overall sorting efficiency was seven recovered single rare cells out of 10 (a fairly typical recovery over many experiments), and the Southern blot revealed that six of those seven sorted cells were MCF-7 cells (lanes A, D, E, F, G, and J), showing that the sort classification was fairly accurate. (Leary, James F. "Ultra High-Speed Sorting", Cytometry Part A 67A:76-85, 2005)
ous transplantation in cancer patients. LEAP has throughputs greater than 100,000 events/sec, while maintaining high cell purity, yield, and viability. It can process several cells or a billion cells with an expanded cell range, including fragile cells. Another advantage is that it can analyze and purify biohazardous cells without generating aerosols.