Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-07-27
2002-07-16
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S001100, C435S006120, C435S091100, C435S091200, C435S270000
Reexamination Certificate
active
06420105
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present application is directed to a single cell preparation method, or a brain cell preparation that provides near-optimal conditions for the accurate quantitation of gene expression in the single cell or neuron. The present invention also provides for a method for studying the functional activity in the same single cell. Also, the method of the present invention relates to the collection of a single neuron, with its membrane and dendritic processes substantially intact and with a full set of mRNA transcripts.
2. Brief Description of the Related Art
With the remarkable explosion in gene expression “chip” technology in recent years it appears that it will soon be possible, perhaps routine, to measure the expression of hundreds or thousands of genes simultaneously, under a wide variety of physiological and pathological conditions. In the foreseeable future, this new technology will clearly overwhelm our capacity to analyze the functional implications of even a fraction of the gene expression patterns that will be found. Taking the central nervous system (CNS) as an example, eventually unraveling the complex linkages between multiple gene expression and CNS function will likely require studies of physiological properties in the same neuron in which multiple gene expression is also assessed.
In turn, such studies will depend on the availability of highly specialized preparations. An optimal CNS preparation for relating gene expression to function in the same neuron should seemingly have three major attributes: 1) the ability to perform extensive functional studies on the same single neuron that is subsequently collected for gene expression analysis; 2) the ability to collect the full set of mRNA transcripts from that same neuron; and 3) the ability to collect the neuron with most of its processes intact, in order to preserve subcellular and dendritic mRNA distribution. However, the main preparations commonly used in brain studies of gene expression, including assays in homogenized tissues (RNAse protection assays, Northerns), in situ hybridization, acutely dissociated neurons, or electrophysiological recording with extraction of cytoplasmic contents (e.g., in non-dissociated slices or cultures), do not provide optimal conditions for parallel measures of function and gene expression in the same neuron. In particular, none of these approaches routinely allows for the collection of the entire cell, including such specialized cells as neurons with their dendritic processes intact. Without a full set of mRNA transcripts, accurate estimates of specific mRNA content are difficult to obtain, and specific dendritically-targeted mRNAs are lost.
Gray et al. discloses a partially-dissociated hippocampal slice (e.g., the “zipper slice” which gradually opens—or unzips—along the cell body layers) in young guinea pigs to provide improved accessibility to neurons for patch clamp pipettes. However, Gray et al. does not disclose the method of the invention whereby a substantially intact single cell is isolated whereby substantially all of the mRNA is detected.
The invention brings together technology encompassing cutting edge instrumentation for electrophysiology, confocal laser scanning microscopy (CLSM), immunoautoradiography and histochemistry, real-time detection of PCR kinetics and new DNA “chip” technology (GeneChip Scanner and Analysis Suite), to optimize and extend the invention.
While there is clearly growing recognition of the value of single cell expression-function studies in the CNS, it seems less well recognized that the preparations that are most widely available for linking function to gene expression in single cells are significantly limited in their usefulness for these purposes. For example, the physiological recording methods in which cytoplasm is extracted through a pipette, do not yield the full complement of the cell's mRNA nor do they allow correlations with topographic mRNA distribution. The proportion of total mRNA extracted varies so greatly from one cell to another that there have been few attempts to estimate the total amount of any mRNA species in a given neuron, or even to estimate relative or semi-quantitative levels. The observations based on this method to date have been generally limited to all-or-none types of findings, regarding, for example, whether or not a gene is expressed in a given cell, or in some cases, whether its ratio of expression to other genes (e.g., for different receptor subunits) is changed (e.g., Sudweeks and Twyman, 1995). In contrast, semi-quantitative or quantitative estimates of the absolute amount of expression of a gene in a cell generally requires collecting the entire complement of a neuron's mRNA transcripts. This seems particularly critical for studies on function-expression correlation in the same cells since the physiological/pathological properties of a cell (e.g., density of channels or receptors, developmental stage, biochemical phenotype or pathological change) may well reflect total level of a gene's expression rather than the ratio to another gene's expression, which might also be altered.
Values obtained with the ratio normalizing approach required when fractional cellular contents are extracted, can be substantially affected by the additional error contributed by variability in the “control” message to which the target message is normalized, by different efficiencies (e.g., in PCR) between the two messages, or by the very common occurrence in which the normalizing message is also up- or down-regulated in tandem, either by the conditions under investigation or by other conditions of the cell (e.g., size, metabolic activity) that affect both messages. The latter may sometimes provide a control for non-specific effects, but in more cases is likely to wash out the absolute value of the target gene signal with which the investigated function may be correlated. Further, it is becoming clear that differential targeting and distribution of mRNAs within the cell (e.g., dendrites or soma) play critical roles in the CNS (Steward et al., 1998; Kuhl and Shehel, 1998). The collection of mRNA by cytoplasmic extraction loses this differential distribution.
Thus, without the ability to obtain the full complement of mRNA with its topographic distribution intact, it will clearly be difficult to estimate the total amount of a mRNA species in a neuron. In turn, this will make it extremely difficult, in most cases, to draw quantitative conclusions about the relations between gene expression and physiological function in individual cells. It should be noted that although the term “gene expression” is used here as a short hand somewhat interchangeably with mRNA content, this is only for purposes of simplicity and it is well recognized that the two are not necessarily equivalent. In the present application, we focus only on the quantitation of total mRNA transcripts, but controls are of course required in many types of experiment before concluding that mRNA content directly reflects expression.
Conventional Methods for mRNA Expression Measurement in the Brain
The main available preparations are: a) Homogenized Tissues: Neuronal and glial heterogeneity generally prevents the accurate assessment of gene/mRNA expression relative to specific cell types or functions, even if small regions of brain are dissected, homogenized, and analyzed by conventional methods (RPAs, Northerns) b) In Situ Hybridization allows visualization of topographic mRNA expression in single cells, but because the tissue is fixed, does not usually permit functional measures (electrophysiology, optical imaging) from the same cells or collection of the mRNA pool for subsequent amplification; also, quantitative analysis (grain counting) is performed by sampling on one section, lending some error to the estimate of total mRNA; c) Acute Dissociation of brain neurons disrupts the membrane and amputates processes. It is not suitable for use with aged or even mature adult neurons as these are often highly trauma
Blalock Eric
Chen Kuey-Chu
Kaminker Patrick
Landfield Philip W.
Thibault Olivier
Horlick Kenneth R.
McDermott & Will & Emery
Spiegler Alexander H.
University of Kentucky Research Foundation
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