Methods for defining cell types

Details Methods for defining cell types Methods for defining cell types

2000-05-09

2002-08-27

Crouch, Deborah (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C435S069100, C435S320100, C435S325000, C435S455000, C800S003000, C800S025000, C800S021000

Reexamination Certificate

active

06441269

ABSTRACT:

INTRODUCTION
1. Field of the Invention
The field of this invention is defining markers for cell types.
2. Background
The identity of a cell is a direct manifestation of the specific complement of genes that it expresses from among the 50,000 to 100,000 genes in the genome. Because individual cell types usually exist to perform specific functions within the organism, a technology that defines cell types through gene expression would not only permit us to assign the expression of genes to functionally defined cell types, but it would also enable us more easily to discover genes imparting functionally relevant properties to individual cells. This assignment of function to gene sequences is a major goal of the field of genomics.
A technology to identify distinct cell types systematically based upon patterns of gene expression would therefore permit very useful, functionally important definitions of cells.
Approaches to such a technology have usually involved performing pairwise comparisons of expressed genes from different cell types (for example, differential display or subtractive hybridization). A shortcoming of such approaches is the impracticality of using pairwise comparisons to identify numerous cell types in a complex tissue. Furthermore, such approaches usually rely upon the ability to isolate cells as pure populations, a situation that does not exist for most cell types in most tissues. Technologies are also needed that would allow the identification of cell types without knowing in advance that they exist. In the human brain, for example, neurons have historically been defined by parameters such as morphology, position, connectivity, and the expression of a small number of marker genes. However, we do not know how many intrinsically different cell types exist in the brain, what functional differences most of these cell types have, and how these differences are manifested in the expression of specific genes. A solution to a problem of this magnitude requires development of new technologies. We describe such a technology here.
Relevant Literature
Sippel (1973) Eur.J.Biochem. 37, 31-40 discloses the characterization of an ATP:RNA adenyltransferase from E. coli and Wittmann et al. (1997) Biochim.Biophys.Acta 1350, 293-305 disclose the characterization of a mammalian poly(A) polymerase. Gething et al. (1980) Nature 287, 301-306 disclose the use of an ATP:RNA adenyltransferase to polyadenylate the '3 termini of total influenza virus RNA. Eberwine et al. (1996) U.S. Pat. No.5,514,545 describes a method for characterizing single cells based on RNA amplification. Eberwine et al. (1992) Proc.Natl.Acad.Sci USA 89, 3010-3014, describe the analysis of gene expression in single live neurons. Gubler U and Hoffman B J. (1983) Gene (2-3), 263-9, describe a method for generating cDNA libraries, see also the more recent reviews, Gubler (1987) Methods in Enzymology, 152, 325-329 and Gubler (1987) Methods in Enzymology, 152, 330-335. Clontech (Palo Alto, Calif.) produces a “Capfinder” cloning kit that uses “GGG” primers against nascent cDNAs capped with reverse transcriptase, Clontechniques 11, 2-3 (Oct. 1996), see also Maleszka et al. (1997) Gene 202, 39-43.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for defining a cell type. The general methods involve the steps of (a) amplifying the mRNA of a single cell of a heterogenous population of cells; (b) probing a comprehensive expression library with the amplified mRNA to define a gross expression profile of the cell; and (c) comparing the gross expression profile of the cell with a gross expression profile of one or more other cells to define a unique expression profile of the cell, wherein the unique expression profile of the cell provides a marker defining the cell type. In particular embodiments, step (c) comprises comparing the gross expression profile of the cell with a gross expression profile of (i) a plurality of other cells to define a unique expression profile of the cell; (ii) a plurality of other single cells to define a unique expression profile of the cell; and/or (iii) a plurality of gross expression profiles of each of a plurality of other single cells to define a unique expression profile of the cell, and the plurality of other single cells are derived from a functionally or structurally distinct subpopulation of cells. Accordingly, the invention may involve the steps of: (a) defining a heterogenous subpopulation of cells of an organism; (b) constructing a comprehensive library from the mRNA of the subpopulation of cells; (c) amplifing the mRNA of a single cell of the population; and (d) probing the library with the amplified mRNA to define gene expression of the cell, wherein the gene expression of the cell provides a marker defining the cell type.
The subpopulation of cells comprises a discernable group of cells sharing a common characteristic. For example, the subpopulation may comprise tissue-specific cells, e.g. hippocampal neurons, cells presenting a common marker, such as CD8+cells, etc. In one embodiment, the marker derives from a common mutation, particularly where the mutation is an inserted genetic construct which encodes and provides each cell with a common selectable marker, such as an epitope or signal-producing protein. In a preferred embodiment, the inserted construct further encodes and provides each cell an internal ribosome entry sequence and the construct is inserted into a target gene downstream of the stop codon but upstream of the polyadenylation signal in the last exon of the target gene, such that the internal ribosome entry sequence provides a second open reading frame within a transcript of the target gene. Selection and/or separation of the target subpopulation may be effected by any convenient method. For example, where the marker is an externally accessible, cell-surface associated protein or other epitope-containing molecule, immuno-adsorption panning techniques or fluorescent immuno-labeling coupled with fluorescence activated cell sorting are conveniently applied.
The probed library is typically a cDNA library, preferably normalized or subtracted.
In a particular embodiment, the library comprises a high density ordered array of immobilized nucleic acids.
The mRNA may be amplified by any technique applicable to a single cell. In a particular embodiment, the amplification is a linear method comprising the steps of adding a known nucleotide sequence to the 3′ end of a first RNA having a known sequence at the 5′ end to form a second RNA and reverse transcribing the second RNA to form a cDNA.
Finally, the library is probed with the amplified mRNA to determine gene expression of the subject cell wherein unique gene expression or gene expression patterns provide markers for defining the cell type.


REFERENCES:
patent: 5514545 (1996-05-01), Eberwine et al.
patent: 5716785 (1998-02-01), Barchas et al.
patent: 91/18115 (1991-11-01), None
patent: 95/08647 (1995-03-01), None
patent: 96/14435 (1996-05-01), None
patent: 97/26333 (1997-07-01), None
Moreadith et. al.; Gene targeting in embryonic stem cells: the new physiology and metabolism, 1997, J. Moi. Med. 75: 208-216.*
Mullins et. al.; Perspectives Series: Molecular Medicine in Genetically Engineered Animals, 1996, J. Clin. Invest. vol. 98, No. 11: S37-S40.*
Shiral; A Gene Trap Strategy for Identifying the Gene Expressed in the Embryonic Nervous System, 1996; Zoological Science 13: 277-283.*
Hansbrough et. al.; A Transgenic Mouse Model for Studying the Lineage Relationships and Differentiation Program of Type 11 Pneumocytes at Various Stages of Lung Development, 1993; Journal of Biological Chemistry, vol. 268:9762-9770.*
Hatada et.al.; The Influence of Chromosomal Location on the Expression of Two Transgenes in Mice, 1999, Journal of Biological Chemistry, vol. 274, No.2: 948-955.*
Eberwine et al., PNAS vol. 89, 1992 pp. 3010-3014 Apr., 1992.
Liu et al., NAR vol. 21 No. 21 pp. pp. 4954-4960 1993.
McDonough et al., EP 0 617 132 A2, Mar.28, 1994.
McDonough et al., N-GeneSeq32, ID No. Q74624, May 5, 1995.

Affiliated with

Also associated with

Rating

Methods for defining cell types does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Methods for defining cell types, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Methods for defining cell types will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2968651