Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-06-04
2004-04-20
Nguyen, Dave Trong (Department: 1632)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C536S023500, C435S320100, C435S325000
Reexamination Certificate
active
06723534
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to isolated and purified proteins and nucleic acids which modulate stem cell renewal, growth and division and which modulate primordial germ cell proliferation. More particularly, the present invention relates to isolated and purified piwi family proteins and isolated and purified polynucleic acids encoding the same.
The publications and other materials used herein to illuminate the background of the invention, and in particular cases, to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and year of publication in the following text, and respectively grouped by author in the appended list of references.
TABLE OF ABBREVIATIONS
APC
apical polar cell(s)
ATCC
American Type Culture Collection
bFGF
basic fibroblast growth factor
BRL
buffalo rat liver
BSA
bovine serum albumin
Gb
cystoblast
CDR(s)
complementarity determining region(s)
COOH
carboxy terminus
CyO
a strain of Drosophila
DAPI
a DNA specific dye
DMEM
Dulbecco's modified Eagle's medium
DC
differentiating 16-cell cysts
dpc
days post coitum
dpp
days post partum
DTC
distal tip cell
ECL
enhanced chemiluminescent
EDTA
ethylenediamine tetraacetate
ES
epithetial sheath
EST
expressed sequence tags
FBS
fetal bovine serum
FITC
fluorescein isothiocyanate
FC
follicle cell
FRT
40A
a strain of Drosophila
Fu
fusome
GC-MS
gas chromatography-mass spectroscopy
Ge
germarium(ia)
GFP
green fluorescent protein
GSC(s)
germline stem cell(s)
HAT
cell culture media comprising hypoxanthine,
aminopterin, and thymidine
hiwi/HIWI
a human homolog of the piwi/PIWI gene
and gene product
HPLC
high pressure liquid chromatography
hsGal4
a heat shock inducible transgene
IS
inner sheath
KLH
keyhole limpet hemocyanin
MC
mitotically active cysts
miwi/MIWI
a murine homolog of the piwi/PIWI gene
and gene product
MmGFP
mutant GFP with improved stability
MPZ
mitotic proliferation zone
myc
human oncogene used herein as molecular
tag for piwi
NH2
amino terminus
ORF
open reading frame
PCR
polymerase chain reaction
PGC(s)
primordial germ cell(s)
piwi/PIWI
a gene and gene product isolated from
Drosophila and having stem cell division
modulating activity
RACE
rapid amplification of cDNA ends
RNAi
RNA interference assay
SSC(s)
somatic stem cell(s)
Sp
spectrosome
TFC
terminal filament cells
TSX
testis-specific X-linked gene product
TUNEL
TdT-mediated dUTP-digoxigenin nick end-
labeling
UTR
untranslated region
VASA
a germline specific marker
WT
wild type
BACKGROUND ART
Stem cells are a very small number of founder cells that play a central role in tissue development and maintenance. In human bodies, stem cells are responsible for generating and/or maintaining approximately 90% of cells in the adult tissues. Over-proliferation of malignant stem cells is the leading cause of cancer while under-proliferation of stem cells or stem-like progenitor cells leads to tissue dystrophy, anemia, immunodeficiency, and male infertility. The crucial role of stem cells has long been attributed to their ability to self-renew and to generate immense number of specialized cells on demand.
The ability of stem cells to self-renew and to produce a large number of differentiated progeny is critical for the development and maintenance of a wide variety of tissues in organisms ranging from insects to mammals (reviewed in Potten, 1997; Lin, 1997; Lin and Schagat, 1997; Morrison et al., 1997). This self-renewing ability is controlled both by extrinsic signaling and by cell-autonomous mechanisms (reviewed in Morrison et al., 1997; Lin and Schagat, 1997). Cell autonomous mechanisms have been elucidated in a few stem cell models such as neuroblasts and germline stem cells in Drosophila (Lin and Schagat, 1997; Deng and Lin, 1997), whereas the role of extrinsic signaling has been elucidated in several systems. For example, the proliferation and differentiation of mammalian stem cells in the hematopoietic, epidermal, and nervous systems depend on extrinsic signals that act on specific receptors on the stem cell surface (Morrison et al., 1997).
In diverse organisms ranging from invertebrates to mammals, the proliferation of germ cells, some of which possess stem cell properties, has been postulated, and, in some cases, shown to be regulated by neighboring non-mitotic somatic cells (Lin, 1997). Particularly, in
C. elegans
, cell—cell interactions between the somatic distal tip cell (DTC) at the end of each gonadal arm and the underlying mitotic germline nuclei via the lag-2lg/p-1 signaling pathway provides a paradigm for soma-germline interaction (reviewed in Kimble and Simpson, 1997). The glp-1 pathway is required to maintain a population of mitotically active nuclei in the germline.
However, few molecules and/or mechanisms identified in a particular type of stem cells have been shown to be applicable to other stem cell systems. For example, the glp-1 equivalent pathway in Drosophila does not play a role in regulating GSC division and maintenance (Ruohala et al., 1991; Xu et al., 1992).
The self-renewing asymmetric division of GSCs in the Drosophila ovary is known to be controlled both by an intracellular mechanism (Deng and Lin, 1997) and by cell—cell interactions (Lin and Spradling, 1993). The intracellular mechanism involves a cytoplasmic organelle termed the spectrosome that controls the orientation of GSC division (Lin et al., 1994; Deng and Lin, 1997). The cell—cell interaction mechanism involves terminal filament cells, as shown by laser ablation studies (Lin and Spradling, 1993). Recently, dpp has been shown as a key signaling molecule required for GSC division and maintenance (Xie and Spradling, 1998). It is possible that the dpp signal emanates from somatic cells. Alternatively, dpp signal may originate from the germline or even within GSCs, like its mammalian homologs (Zhao et al., 1996).
In mammals, primordial germ cells cultured from the genital ridge have the ability to give rise to pluripotent embryonic stem cells. For example, U.S. Pat. No. 5,690,926 issued Nov. 25, 1997 to Hogan; U.S. Pat. No. 5,670,372 issued Sep. 23, 1997 to Hogan; and U.S. Pat. No. 5,537,357 issued Sep. 26, 1995 to Hogan each disclose pluripotential mammalian embryonic stem cells and methods of making the same. The disclosure of these patents is limited to mammalian embryonic stem cells and particularly to the culturing of murine and other mammalian embryonic stem cells using a combination of growth factors consisting of SCF, FGF and LIF.
Current prior art reports on the culture of avian primordial germ cells (PGCs) have concentrated on efforts to maintain a PGC-phenotype and to stimulate proliferation. See e.g., Chang, I. K. et al.,
Cell. Biol. Int.
1997 August; 21(8): 495-9; Chang, I. K. et al.,
Cell. Biol. Int.
1995 February; 19(2): 143-9; Allioli, N. et al.,
Dev. Biol.
1994 September; 165(1): 30-7 and PCT Publication No. WO 99/06533, published Feb. 11, 1999 (Applicant—University of Massachusetts; Inventors—Ponce de Leon et al.).
As illustrated above, numerous attempts have been devoted to identify genes that control the self-renewing ability of stem cells or the proliferation of primordial germ cells. As a result, a number of growth factors and signaling molecules, such as Steel factor and its c-kit receptor, have been identified to regulate such activity in certain tissues. Despite this progress, there remains a long-felt and continuing need to identify genes that play a role in modulating the growth and self-renewing division of stem cells, particularly GSCs, and that play a role in modulating proliferation of primordial germ cells.
SUMMARY OF THE INVENTION
The present invention contemplates an isolated and purified family of genes and gene products (the piwi family) which plays a role in the growth, proliferation and self-renewing division of stem cells, and proliferation of primordial germ cells. More preferably, a polypeptide of the invention is a recombinant polypeptide. Even more preferably, a polypeptide of the present invention comprises a vertebrate piwi
Duke University
Jenkins & Wilson & Taylor, P.A.
Nguyen Dave Trong
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