Performing Department
(N/A)
Non Technical Summary
Project Summary:We aim to procure a flexible and user-friendly laser microdissection (LMD) system to bolster current food and agricultural research and elevate capabilities in the North Texas and Southern Oklahoma region. The equipment will be housed at the University of North Texas. LMD offers proven, versatile technology for precision sample preparation: it is the gold standard for obtaining well-defined, discrete materials for downstream experiments. In biological, agricultural, and biomedical sciences, isolating specific organs, tissues, or individual cells--with microscopic resolution--facilitates the finest scale of discovery and is particularly important for developmental biology, biochemistry, pathology, and systems biology related "-omics" technologies. LMD at UNT will help reclaim regional capabilities that were lost with the dissolution of basic sciences at the Noble Research Institute in Ardmore, OK. A strength of our proposal is how well LMD will complement and augment existing facilities: UNT's Life Sciences Complex houses state-of-the-art microscopy and histology resources for upstream tissue preparation and is well-equipped for cutting-edge downstream analyses with the UNT Genomics Center and the BioAnalytical Facility for metabolomics; coupled with UNT's Laboratory for Imaging Mass Spectrometry, LMD will provide a powerful technique to isolate and analyze tissues showing distinctive metabolite signatures. Regional institutions with strengths in food and agricultural research that will benefit from LMD capabilities, as described in this proposal, include UNT and Texas Woman's University, both Hispanic- and Minority-Serving Institutions in Denton, TX; Texas A&M University AgriLife at Dallas; and the Oklahoma State University Institute for Agricultural Biosciences in Ardmore, OK.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
We will procure a flexible and user-friendly laser microdissection (LMD) system to bolster current food and agricultural research and elevate capabilities in the North Texas and Southern Oklahoma region. The equipment will be housed at the University of North Texas. LMD offers proven, versatile technology for precision sample preparation: it is the gold standard for obtaining well-defined, discrete materials for downstream experiments. In biological, agricultural, and biomedical sciences, isolating specific organs, tissues, or individual cells--with microscopic resolution--facilitates the finest scale of discovery and is particularly important for developmental biology, biochemistry, pathology, and systems biology related "-omics" technologies. LMD at UNT will help reclaim regional capabilities that were lost with the dissolution of basic sciences at the Noble Research Institute in Ardmore, OK. A strength of our proposal is how well LMD will complement and augment existing facilities: UNT's Life Sciences Complex houses state-of-the-art microscopy and histology resources for upstream tissue preparation and is well-equipped for cutting-edge downstream analyses with the UNT Genomics Center and the BioAnalytical Facility for metabolomics; coupled with UNT's Laboratory for Imaging Mass Spectrometry, LMD will provide a powerful technique to isolate and analyze tissues showing distinctive metabolite signatures. Regional institutions with strengths in food and agricultural research that will benefit from LMD capabilities, as described in this proposal, include UNT and Texas Woman's University, both Hispanic- and Minority-Serving Institutions in Denton, TX; Texas A&M University AgriLife at Dallas; and the Oklahoma State University Institute for Agricultural Biosciences in Ardmore, OK.Brian Ayre (Prof., BDI, and Dept. Biol. Sci., UNT). I study plant vascular systems.I hypothesize that there are progressive cascades of developmental and biosynthetic genes expressed as cells are pushed further from the cambia and differentiate toward their final fate. A specific hypothesis that LMD will help us test is that the signaling pathway(s) leading to phloem cap fibers is related to, and possibly a localized reiteration of, the pathways contributing to wood formation on the xylem-side of the cambia.Roisin McGarry (Res. Assoc. Prof., BDI and Dept. Biol. Sci., UNT). I investigate meristem homeostasis and plant architecture and by unraveling and leveraging the genetic networks regulating meristem fate and size, I propose that plant architecture can be modified to increase productivity.Applying LMD to fixed meristems will vastly improve the resolution and homogeneity of isolated tissues and will enable my students and I to build more robust models describing how organ-specific genetic networks contribute to the phenotype of the mature fruit.Jyoti Shah (Prof., BDI and Chair, Dept. Biol. Sci., UNT). My group studies the molecular and physiological bases of plant-aphid interactions. A prominent trajectory is to identify defense genes induced at the site of stylet penetration into the phloem and to understand how they affect molecular events and host physiology. LMD, combined with our existing genetic resources and electrophysiology techniques [37], will allow cell sampling at the site of insect feeding from control and transgenic experimental plants, followed by analyses of transcriptome, proteome, and metabolome changes.Vanessa Macias (Assist. Prof., Dept. Biol. Sci., UNT). I study PIWI-interacting RNA (piRNA) in heterogenous insect tissues as a heritable genetic immunity to foreign nucleic acids, such as viruses and synthetic transgenes.I hypothesize that a stepwise interplay between RNAi and maternal follicle cells is followed by piRNA-mediated communication with the oocytes. Further, we have observed that transgenes appear differentially regulated between cells within the same tissue and piRNA-mediated regulation may be responsible. LMD paired with piRNA enrichment and sequencing will allow us to characterize distinct small RNA populations and functionally validate our hypotheses.Patrick Horn (Assist. Prof., BDI and Dept. Biol Sci., UNT). I study redox biochemistry and plant lipid metabolism. My collaborators and I hypothesize that if we can identify genes contributing to the biosynthesis and compartmentation of cyclic fatty acids (CFAs), we will be able to engineer targeted bioproduct compositions in cotton and ultimately other crops. CFAs are rare, unusual FAs with potential value in plant defense as well as nutritional and industrial feedstocks.
Project Methods
The users of the laser microdissection facility have a broad diversity of research interests and will similarly have a diversity of methods.LMD offers proven, versatile technology for precision sample preparation: it is the gold standard for obtaining well-defined, discrete materials for downstream experiments. In biological, agricultural, and biomedical sciences, isolating specific organs, tissues, or individual cells--with microscopic resolution--facilitates the finest scale of discovery and is particularly important for developmental biology, biochemistry, pathology, and systems biology related "-omics" technologies.LMD equipment is intuitive and users with basic microscopy skills can cut and collect samples.LMD instrumentation consists of a programmable fluorescence microscope with a robotic stage, a diode-pulsed solid-state laser for cutting, a mechanism to collect excised samples, cameras for image capture, and associated computer hardware and software (see attached quote). Generally, cryo-sectioned tissues are preferred for isolating biologically active components such as DNA, RNA, proteins, and metabolitesbut fixed and paraffin-embedded sections can be used, and there are also techniques for excision from living cells. From these starting materials, multiple sections are adhered to a single slide and visualized; cells of interest are traced using a touchpad and the excision and collection of the outlined cells proceeds at the touch of a button. The automated stage can be programmed to cut sequentially through a Z-series to permit isolation from thick sections, and the adjustable laser is strong enough to cut bone samples and tough plant cell walls, or gentle enough to cut soft tissues without damage. Commercial kits to process DNA and RNA at the scale of single cells are available from several sources (New England Biolabs, Takara Bio, Illumina, etc.) such that the quantity of tissue required for analysis is continually being reduced so investigators can focus on sample purity.We are purchasing the Leica LMD7. The LMD7 has higher laser power and more laser controls than other models, making it ideally suited to the diverse needs of a shared-use facility. Unique to Leica is that sample collection is by gravity: once excised, the sample simply falls from the slide into a collection vessel and avoids overly complicated 'catapulting' mechanisms or adhesion to proprietary collection caps. This facilitates collecting samples into standard PCR tubes, strips, or microtiter plates--dry or prefilled with, e.g., RNA isolation solution--which substantially reduces the cost of consumables.