A Cryosectioning and Laser Microdissection Facility for Gene Metabolite and Protein Profiling from Pure Populations of Cells and Cell Types

Lead Research Organisation: Rothamsted Research
Department Name: Plant Genetics and Breeding

Abstract

Laser microdissection (LMD) is a relatively new technique that is revolutionising the way scientists analyse biological processes. It allows researchers to examine a specimen under the microscope, cut out a precise area of interest (a single cell, perhaps, or a collection of cells) with a laser, and then collect the excised sample in a tube without contaminating it in any way. Once the sample has been captured, it can be subjected to a wide spectrum of chemical analyses as required. These can include analyses of DNA, RNA, proteins and the thousands of other cell chemicals that are the foundation of biological processes. In the case of many chemicals (DNA and RNA, for example) routine methods now allow researchers to work with the tiny amounts of these substances that can be isolated from single cells. Although all cell types share their basic chemistry, it is the subtle differences between them that give them their unique character and functions. They express different genes, build different proteins and contain different suites of active molecules. Understanding these differences and their purpose is central to many areas of the biological sciences. Without the LMD technique, researchers often have to work with samples containing many different types of cells. This means that the unique character of individual cell types is difficult or impossible to discern, because it is masked by the presence of several types of cell. As a result, it is often difficult to study specific localised events such as cell death, fungal infection or transport of proteins or sugars in or out of specific cells. Although single cells can be sampled with a micro needle if they are easily accessible, many cells are hidden within tissues and cannot be reached directly. LMD answers these problems by allowing easy access to any part of any material that can be cut into thin slices and mounted on a microscope slide. Because the slices are prepared from quickly frozen tissue (using a supplementary piece of equipment, a freezing microtome), cells suffer the absolute minimum of disturbance before examination and laser dissection. Access to these cells via LMD will help IGER researchers to understand key biological processes with greater precision. IGER carries out a wide range of research designed to underpin the targeted development of new plant varieties and agricultural methods, which are better for farming and better for the environment. Other things being equal, increased precision in research will accelerate the pace of discovery and development. Many research projects will benefit directly from the new equipment. These include studies aimed at understanding the roles of specific genes in plant growth and development, studies on plant responses to stress, and studies on the interactions taking place when plant material is broken down in the rumen of farm animals. These research programmes are central to key aims of UK science, including development of renewable energy for biofuels and the development of sustainable agriculture. LMD will be used to analyse gene expression in specific plant cells, focusing on areas such as plant senescence following stress. It will contribute to ongoing studies that are examining the way different types of cell process carbohydrate in the leaves of grasses. Studies on signalling between plant cells during fungal invasion will also benefit. LMD will help researchers study colonisation of plant material by microorganisms in the rumen, a key part of digestion in ruminant animals. Specifically, there are plans to follow gene expression during colonisation and to understand what microbial species are involved.

Technical Summary

Laser-assisted microdissection (LMD) techniques offer unprecedented access to specific cells and cell types for defining patterns of gene expression in combination with powerful technologies, such as gene array and real-time PCR. We will apply existing techniques to correlate expression profiles of genes and proteins in samples ranging from single cells to populations of thousands of cells isolated from specific tissues of a range of plant species, including grasses and clovers. We will isolate populations of pure single cells and tissue samples from shoot, root and floral tissues of plants and analyse expression of genes involved in resource (carbohydrate) allocation, biofuel production, root and nodule senescence, protein protection, plant fertility and signal transduction. Expression patterns of such genes will be correlated with protein expression by proteomics and by developing techniques for analysing specific protein activity in small tissue samples, using a NanoDrop ND-1000 spectrophotometer. We will also extend the techniques to establish metabolite profiling within specific cell types using FTIR metabolite fingerprinting and profiling with GC-MS and LC-MS. LMD offers a powerful tool for understanding the functioning of the animal rumen and dissecting its microbial community. We will investigate gene activity in infected and non-infected plant tissues in the rumen, conducting differential display and micro-array approaches to investigate localised changes in gene expression as a response to invading pathogens. We will construct a microbial profile of excised, colonised regions of plant tissues (both internal and cut surfaces) in the rumen using existing bacterial primers. In addition, we will develop LDM live cell cutting under high CO2 conditions to confirm the identity of sporangia of obligate aerobic fungi, using a combination of DNA profiling and the morphology of the excised surviving fungus.

Publications

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