Method of determining multiple mRNAs in dying cells

Details Method of determining multiple mRNAs in dying cells Method of determining multiple mRNAs in dying cells

1999-03-23

2001-12-04

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200

Reexamination Certificate

active

06326146

ABSTRACT:

BACKGROUND OF THE INVENTION
The ability to assess the expression of multiple genes in individual cells represents a powerful tool for studying the messenger RNA (mRNA) abundances within identifiable cell types. One such technique, the amplified antisense RNA (aRNA) method (Eberwine, J. et al. 1992
Proc. Natl. Acad. Sci. USA
89:3010-3014) allows the simultaneous identification of relative messenger RNA (mRNA) levels for multiple genes within single cells. The first introduction of in situ transcription (IST) allowed the analysis of gene expression within fixed tissue sections (Tecott, L. H. et al. 1988 Science 240:1661-1664). The recent development of the aRNA procedure coupled with IST permitted the analysis of the relative levels of multiple mRNAs within single, dissociated cells (Eberwine, J. et al. 1992
Proc. Natl. Acad. Sci. USA
89:3010-3014). In addition, the aRNA technique can be combined with electrophysiological recordings from dissociated cells (Eberwine, J. et al. 1992
Proc. Natl. Acad. Sci. USA
89:3010-3014) or cells from slice preparations (Mackler, S. A. et al. 1992 Neuron 9:539-548) to provide a functional correlate of gene expression changes. The cellular specificity of aRNA amplification offers a distinct advantage over other techniques used to evaluate gene expression. For example, Northern analysis involves extraction of RNA from tissue homogenates which include a heterogeneous population of cells particularly within the central nervous system (CNS). In situ hybridization can be used to study gene expression in individual cells, but the study of multiple genes is complex. Another method, PCR, limits analysis to only a few genes at one time (Eberwine, J. et al. 1995
The Neuroscientist
1:200-211).
Recently, the aRNA procedure has been extended to characterize the expression of mRNA abundances for multiple genes within immunohistochemically labeled cells (Crino, P. B. et al. 1996
Proc. Natl. Acad. Sci. USA
93:14152-14157). This method allows additional phenotypic characterization of cells prior to single cell amplification. While this method of cell identification is useful for identifying cells based on the expression of a particular protein, imamunohistochemical detection is problematic when applied to analyzing the molecular changes in degenerating or dying cells. Specifically, a frequent hallmark of damaged cells is disruption of protein turnover. Therefore, particular proteins may be decreased or even absent in dying cells. Such alterations in protein expression and activity have been reported for a variety of CNS insults (Ferrer, I. et al. 1993
Clin. Neuropath
. 12:53-58; Taft, W. C. et al. 1993
J. Cereb. Blood Flow Metab
. 13:796-802; Hicks, R. R. et al. 1996
Acta Neuropathol
. 91:236-246). However, other proteins are upregulated in neurologic disease (Anderson, A. J. et al. 1994
Exp. Neurol
. 125:286-295). Thus, the expression of immunohistochemical markers may reveal abnormal cell populations; or alternatively, the expression of certain proteins in cells may be associated with cell death. Without a specific marker of cell damage, however, positive identification of damaged/dying cells based on immunohistochemical criteria is unreliable.
Methods for identifying dying cells by DNA damage stains have been described. For example, terminal deoxynucleotidyl-transferase (Tdt) mediated biotin-dUTP nick end labeling (TUNEL) technique has been used to identify dying cells in a developing brain (Gavrieli et al. 1992
J. Cell Biol
. 119:493-501). The TUNEL stain utilizes the enzyme Tdt which incorporates biotinylated nucleotides to the 3′ ends of fragmented DNA and has been used as a marker for dying cells. This method is useful for identifying cells that are undergoing programmed cell death (PCD), a phenomenon which occurs as a consequence of normal development (Oppenheim, R. W. 1991
Annual Review of Neuroscience
14:453-501). TUNEL-positive cells have also been found in pathological conditions including traumatic brain injury (Rink, A. et al. 1995
Am. J. Pathol
. 147:1575-1583; Colicos, M. A. and Dash, P. K. 1996
Brain Research
739:102-131; Conti, A. C. et al. 1996
J. Neurotrauma
13:595), ischemia (Li, Y. et al. 1995
Stroke
26:1252-1257), tumors (Ikeda, H. et al. 1996
Am. J. Surg. Pathol
. 20:649-655), Alzheimer's disease (Smale, G. et al. 1995
Exp. Neurol
. 133:225-230; Tronsco, J. C. et al. 1996
J. Neuropathol. Exp. Neurol
. 55:1134-1142), Parkinson's disease (Mochizuki, H. et al. 1997
J. Neural. Transm. Suppl
. 50:125-140), Huntington's disease (Thomas, L. B. et al. 1995
Exp. Neurol
. 133:265-272), multiple sclerosis (Dowling, P. et al. 1996
J. Exp. Med
. 184:1513-1518) and amyotrophic lateral sclerosis (ALS); Troost, D. et al., 1995
Neuropathol. and Applied Neurobiol
. 21:498-504.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for determining expression levels of multiple mRNAs in cells undergoing cell death which comprises detecting dying cells in a selected tissue by a DNA damage stain, preferably by a terminal deoxynucleotidyl-transferase mediated biotin-dUTP nick end labeling technique; isolating single, dying cells or portions thereof; and detecting expression levels in these cells or portions thereof by an amplified antisense RNA technique so that expression levels of multiple mRNAs in these cells or portions thereof are determined.
Another object of the present invention is to provide a method of diagnosing diseases of the central nervous system which comprises detecting dying cells in central nervous system tissue by a DNA damage stain, preferably by a terminal deoxynucleotidyl-transferase mediated biotin-dUTP nick end labeling technique; isolating single, dying cells or portions thereof from the tissue; detecting mRNA expression patterns in these cells or portions thereof by an amplified antisense RNA technique; and correlating the pattern of mRNA expression in the dying cells or portions thereof to a disease of the central nervous system.
Another object of the present invention is to provide a method of evaluating therapeutic strategies in diseases of the central nervous system comprising detecting dying cells in central nervous system tissue by a terminal deoxynucleotidyl-transferase mediated biotin-dUTP nick end labeling technique; isolating single, dying cells or portions thereof from the tissue; detecting mRNA expression patterns in these cells or portions thereof by an amplified antisense RNA technique; identifying mRNAs of the expression pattern which are altered; administering agents expected to compensate for alterations in the identified mRNAs; and evaluating the effects of the administered agent on the central nervous system disease.
DETAILED DESCRIPTION OF THE INVENTION
Trauma to the central nervous system (CNS) is frequent in industrialized countries, affecting many patients in the prime of life. The wide variety of diseases of the CNS that involve cell injury and cell death makes understanding the mechanisms behind brain injury a critical element of clinical research. Further, brain trauma is becoming a larger problem for physicians due to advances in emergency medicine that have led to increased survival of patients with injury to the brain and/or spinal cord.
Methods available for studying the changes that occur during development of CNS trauma, or CNS disease that involves cell death, are limited. Much of the focus has been on methods to prevent cell death. Thus, only limited information is available on the physiological changes that occur during CNS cell death and the cellular mediators of cell death. In the present invention, a method for examining the alterations in mRNA expression levels, also referred to herein as expression patterns or profiles, that occur during cell death in single cells is presented. The method involves aRNA amplification techniques for use in cells identified as dying cells by a DNA damage stain, preferably by a terminal deoxynucleotidyl-transferase (Tdt) mediated biotin-dUTP nick end labeling (TUNEL) technique or other m

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