Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Primate cell – per se
Reexamination Certificate
1997-01-23
2001-06-12
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Primate cell, per se
C435S325000, C435S366000
Reexamination Certificate
active
06245564
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to a method of separating cells of interest from organs and from larger populations of mixed cell types. In particular, this invention includes the introduction of a nucleic acid molecule encoding a green fluorescent protein, under the control of a cell-specific promoter, into a plurality of cells and then separating the cells of interest by detecting fluorescence in those cells in which the cell-specific promoter drives expression of the green fluorescent protein.
BACKGROUND OF THE INVENTION
Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The damaged brain is largely incapable of functionally significant structural self-repair. This is due in part to the apparent failure of the mature brain to generate new neurons (Korr, 1980; Sturrock, 1982). However, the absence of neuronal production in the adult vertebrate forebrain appears to reflect not a lack of appropriate neuronal precursors, but rather their tonic inhibition and/or lack of post-mitotic trophic and migratory support. Converging lines of evidence now support the contention that neuronal precursor cells are distributed widely throughout the ventricular subependyma of the adult vertebrate forebrain, persisting across a wide range of species groups (Goldman and Nottebohm, 1983; Reynolds and Weiss, 1992; Richards et al., 1992; Kirschenbaum et al., 1994; Kirschenbaum and Goldman, 1995a; reviewed in Goldman, 1995; Goldman, 1997; and Gage et al., 1995). Most studies have found that the principal source of these precursors is the ventricular zone (Goldman and Nottebohm, 1983; Goldman, 1990; Goldman et al., 1992; Lois and Alvarez-Buylla, 1993; Morshead et al., 1994; Kirschenbaum et al., 1994; Kirschenbaum and Goldman, 1995), though competent neural precursors have been obtained from parenchymal sites as well (Richards et al., 1992; Palmer et al., 1996; Pincus et al., 1996). In general, adult progenitors respond to epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) with proliferative expansion (Reynolds and Weiss, 1992; Kilpatrick and Bartlett, 1995), may be multipotential (Vescovi et al., 1993; Goldman et al., 1996), and persist throughout life (Goldman et al., 1996). In rodents and humans, their neuronal daughter cells can be supported by brain-derived neurotrophic factor (BDNF) (Kirschenbaum and Goldman, 1995a), and become fully functional in vitro (Kirschenbaum et al., 1994), like their avian counterparts (Goldman and Nedergaard, 1992; Pincus et al., 1996). In general, residual neural precursors are widely distributed geographically, but continue to generate surviving neurons only in selected regions; in most instances, they appear to become vestigial (Morshead and van der Kooy, 1992), at least in part because of the loss of permissive signals for daughter cell migration and survival in the adult parenchymal environment.
A major impediment to both the analysis of the biology of adult neuronal precursors, and to their use in engraftment and transplantation studies, has been their relative scarcity in adult brain tissue, and their consequent low yield when harvested by enzymatic dissociation and purification techniques. As a result, attempts at either manipulating single adult-derived precursors or enriching them for therapeutic replacement have been difficult. The few reported successes at harvesting these cells from dissociates of adult brain, whether using avian (Goldman et al., 1992; 1996c), murine (Reynolds and Weiss, 1992), or human (Kirschenbaum et al., 1994) tissue, have all reported <1% cell survival. Thus, several groups have taken the approach of raising lines derived from single isolated precursors, continuously exposed to mitogens in serum-free suspension culture (Reynolds and Weiss, 1992; Morshead et al., 1994; Palmer et al., 1995). As a result, however, many of the basic studies of differentiation and growth control in the neural precursor population have been based upon small numbers of founder cells, passaged greatly over prolonged periods of time at high split ratios, under constant mitogenic stimulation. The phenotypic potential, transformation state and karyotype of these cells are all uncertain; after repetitive passage, it is unclear whether such precursor lines remain biologically representative of their parental precursors, or instead become transformants with perturbed growth and lineage control.
A strong need therefore exists for a new strategy for isolating and enriching native neuronal precursors and neural stem cells from adult brain tissue. Such isolated, enriched native precursors can be used in engraftment and transplantation, as well as for studies of growth control and functional integration.
SUMMARY OF INVENTION
To this end, the subject invention provides a method of separating cells of interest from a larger, heterogeneous cell population, based upon cell type-selective expression of cell specific promoters. This method comprises: determining the cells of interest; selecting a promoter specific for the cells of interest; introducing a nucleic acid molecule encoding green fluorescent protein under control of the promoter into a plurality of cells; and separating cells of the plurality of cells that are expressing said green fluorescent protein, wherein the separated cells are the cells of interest.
The cells of interest, in a preferred embodiment of the method of the subject invention, are neuronal cells, and particularly neuronal precursor cells. A promoter is chosen which specifically drives expression in the cells of interest, i.e. the promoter drives expression in neuronal precursor cells but not in other cells of the nervous system. The green fluorescent protein will therefore only be expressed and detectable in cells in which the promoter operates, i.e. those cells for which the promoter is specific.
The method involves the introduction of nucleic acid encoding the green fluorescent protein, under the control of the cell specific promoter, into a plurality of cells. Various methods of introduction known to those of ordinary skill in the art can be utilized, including (but not limited to) viral mediated transformation (e.g. adenovirus mediated transformation), electroporation, biolistic transformation, and liposomal mediated transformation.
After cell specific expression of the green fluorescent protein (GFP), the cells expressing the green fluorescent protein are separated by any appropriate means. For example, the cells can be separated by fluorescence activated cell sorting. The method of the subject invention thus provides for the enrichment and separation of the cells of interest.
A presently preferred embodiment of the method of the subject invention relates to neuronal precursor cells which are widespread in the forebrain ventricular zone (VZ), and which may provide a cellular substrate for brain repair. Contemporary approaches toward the use of neuronal precursor cells have focused upon preparing clonal lines derived from single progenitors. However, such propagated lines can become progressively less representative of their parental precursors with time and passage in vitro. To circumvent these difficulties, the method of the subject invention provides a strategy for the live cell identification, isolation and enrichment of native precursors and their neuronal daughter cells, by fluorescence-activated cell sorting of VZ cells transfected with green fluorescent protein, driven by the neuronal T&agr;1 tubulin promoter. Using this approach, neural precursors and their young neuronal daughters can be identified and selectively harvested from a wide variety of samples, including embryonic and adult brain of both avian and mammalian origin.
Extension of this approach to include fluorescent transgenes under the control of stage- and phenotype-specific promot
Goldman Steven A.
Wu Hong
Allen Marianne P.
Cornell Research Foundation Inc.
Nixon & Peabody LLP
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