EP0695351B1
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EP
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Isolation, selection and propagation of animal transgenic stem cells
Assignee: UNIVERSITY COURT OF
•
Filing: 1994-04-21
•
Granted: 1999-12-08
•
Expires: 2014-04-21
Abstract
Abstract Animal stem cells are obtained and maintained by culturing cells containing, in the genome, a selectable marker. Differential expression of the selectable marker enables preferential survival and/or division of the desired stem cells compared to the non-stem cells.
Inventors
- 👤 Austin Gerard Afrc Centre Genome Research Smith
- 👤 Peter Scott Stem Cell Sciences Mountford
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-03 10:39
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/87; C12N15/90; C12N15/902; C12N15/907; A; A01; A01K; A01K67/00; A01K67/027; A01K67/0275; C07; C07K; C07K14/00; C07K14/435; C07K14/46; C07K14/47; C07K14/4701; C07K14/4702; C07K14/52; C07K14/705; C07K14/70596; C07K14/81; C12N15/63; C12N15/79; C12N15/85; C12N15/8509; C12N15/873; C12N5/00; C12N5/06; C12N5/0602; C12N5/0618; C12N5/0623; C12N9/00; C12N9/10; C12N9/12; A01K2217/00; A01K2217/05; A01K2217/07; A01K2217/072; A01K2227/00; A01K2227/10; A01K2227/105; A01K2267/00; A01K2267/02; A61; A61K; A61K38/00; C12N2506/00; C12N2506/02; C12N2510/00; C12N2830/00; C12N2830/008; C12N2830/42; C12N2830/80; C12N2830/85; C12N2840/00; C12N2840/20; C12N2840/203; C12N2840/44
Claims Analysis
Total Claims: 48
Claim 1
Independent Claim
93 words
A method of isolating and/or enriching and/or selectively propagating desired animal
stem cells, which comprises maintaining a source of said cells under culture conditions
conducive to cell survival, characterised in that the source of cells includes stem cells
containing a selectable marker which is capable of differential expression in (a) desired stem
cells of said source and (b) cells of said source other than the desired stem cells, whereby
differential expression of said selectable marker results in preferential isolation and/or survival
and/or division of the desired stem cells containing the said selectable marker.
Claim 2
Dependent Claim
39 words
A method according to Claim 1 wherein the desired stem cells are selected from
unipotential stem cells, pluripotential stem cells, embryonic stem cells, gonadal stem cells,
somatic stem/progenitor cells, haematopoietic stem cells, epidermal stem cells and neuronal
stem cells.
Claim 3
Independent Claim
38 words
A method according to any preceding claim wherein the source of cells includes stem
cells having a positive selectable marker and expression of said marker is used to permit
isolation and/or enrichment and/or maintenance of the stem cells.
Claim 4
Independent Claim
39 words
A method according to any preceding claim wherein expression of a negative
selectable marker in cells other than the desired stem cells is used to selectively deplete the
source of cells of cells other than the desired stem cells.
Claim 5
Independent Claim
25 words
A method according to any preceding claim wherein the selectable marker is selected
from a foreign gene, a cellular gene and an antibiotic resistance gene.
Claim 6
Dependent Claim
17 words
A method according to Claim 5 wherein the antibiotic resistance gene is the bacterial
neomycin resistance gene.
Claim 7
Independent Claim
19 words
A method according to any of Claims 1 to 5 wherein the selectable marker is a growth
stimulating gene.
Claim 8
Dependent Claim
17 words
A method according to Claim 7 wherein the growth stimulating gene is an oncogene
or derivatives thereof.
Claim 9
Dependent Claim
24 words
A method according to Claim 7 wherein the growth stimulating gene is SV40 large
T antigen or a derivative of SV40 large T antigen.
Claim 10
Dependent Claim
49 words
A method according to Claim 7 wherein the growth stimulating gene is selected from
a gene that codes for a growth factor, a gene that codes for a growth factor receptor, a gene
that codes for a signal transducing molecule, and a gene that codes for a transcription factor.
Claim 11
Independent Claim
18 words
A method according to any of Claims 1 to 5 wherein the selectable marker is an
immortalising gene.
Claim 12
Dependent Claim
28 words
A method according to Claim 11 wherein the immortalising gene is selected from the
polyoma large T gene, a gene that blocks cell death and the bcl-2 gene.
Claim 13
Independent Claim
74 words
A method according to any of Claims 1 to 5 wherein the isolation and/or enrichment
and/or propagation of the desired pluripotential cells is dependent on the presence of cells
other than the desired pluripotential cells and the simultaneous maintenance of both cell types
is dependent on expression of a selectable marker, in one or the other cell population, which
is capable of rescuing cells neighbouring those cells which do not themselves express the
marker.
Claim 14
Dependent Claim
43 words
A method according to Claim 13 wherein the selectable marker is selected from
HPRT, a gene encoding a toxic product, a toxic gene product which is conditionally active
in combination with a suicide substrate and a herpes simplex virus thymidine kinase (HSV-TK)
gene.
Claim 15
Independent Claim
14 words
A method according to any preceding claim wherein the cells contain two selectable
markers.
Claim 16
Independent Claim
32 words
A method according to any preceding claim wherein expression of the selectable
marker is achieved by operatively inserting the selectable marker into an expression construct
prior to introduction to the cell source.
Claim 17
Independent Claim
27 words
A method according to any preceding claim wherein expression of the selectable
marker results from the introduction of a stably integrated, episomally maintained or
transiently maintained construct.
Claim 18
Independent Claim
28 words
A method according to any preceding claim wherein expression of the selectable
marker results from operatively inserting the selectable marker into an endogenous gene of
the cell source.
Claim 19
Independent Claim
29 words
A method according to any preceding claim wherein the selectable marker is
introduced into the cells by transfection, lipofection, injection, ballistic missile, viral vector,
electroporation or any other means.
Claim 20
Independent Claim
57 words
A method according to any preceding claim wherein the source of cells is obtained
by culture of a single cell or cell line; cells derived from a fertilized oocyte, or a transgenic
animal or a non-transgenic animal; cells derived from an embryo, from blood or from
somatic tissue; or a mixture of any of the aforementioned cells.
Claim 21
Dependent Claim
25 words
A method according to Claim 20 wherein the source of cells is obtained by culture of
a cell or cells obtained from a transgenic animal.
Claim 22
Independent Claim
35 words
A method according to any preceding claim wherein the selectable marker is
operatively linked to a gene or gene fragment regulating expression which gene or gene
fragment is differentially active in stem and non-stem cells.
Claim 23
Independent Claim
32 words
A method according to any preceding claim wherein a gene or gene fragment
operatively linked to and regulating expression of the selectable marker is/are associated with
a pluripotential stage of cellular development.
Claim 24
Dependent Claim
21 words
A method according to Claim 23 wherein the gene or gene fragment is active in
pluripotential cells of the developing embryo.
Claim 25
Dependent Claim
19 words
A method according to Claim 23 or 24 wherein the gene or gene fragment is active
in primitive ectoderm.
Claim 26
Independent Claim
22 words
A method according to any of Claims 22-25 wherein the gene or gene fragment is all
or part of the Oct4 gene.
Claim 27
Independent Claim
18 words
A method according to any of claims 22-26 wherein the gene or gene fragment is the
Oct4 promoter.
Claim 28
Independent Claim
16 words
A method according to any preceding claim wherein the selectable marker is the
neomycin phosphotransferase gene.
Claim 29
Independent Claim
20 words
A method according to any of Claims 22-28 wherein the gene or gene fragment is
active in pluripotent haematopoietic cells.
Claim 30
Dependent Claim
20 words
A method according to Claim 29 wherein the gene or gene fragment is all or part of
the CD34 gene.
Claim 31
Independent Claim
44 words
A method according to any preceding claim which includes the step of introducing into
a source of cells containing stem cells, a selectable marker construct, wherein said selectable
marker construct is adapted to operatively link to an endogenous gene which provides said
differential expression.
Claim 32
Independent Claim
50 words
A method according to any of Claims 1 to 30 which includes the step of introducing
into a source of cells containing stem cells, a selectable marker construct, wherein said
selectable marker construct has been previously linked to one or more genes or gene
fragments which provide said differential expression.
Claim 33
Independent Claim
92 words
A method of selectively isolating and/or enriching and/or propagating desired animal
stem cells, which comprises introducing into a source of cells containing stem cells, a
selectable marker construct which operatively links to or has been previously linked to genes
or gene fragments which provide differential expression of the selectable marker in (a) desired
stem cells of said source and (b) cells of said source other than the desired stem cells and
which under appropriate culture conditions enables selective isolation and/or enrichment
and/or propagation of the desired stem cells containing the selectable marker.
Claim 34
Dependent Claim
34 words
A method according to Claim 33 wherein the selectable marker is operatively linked
to a gene or gene fragment regulating expression, which gene or gene fragment is
differentially active in stem and non-stem cells.
Claim 35
Dependent Claim
16 words
A method according to Claim 34 wherein the gene or gene fragment is the Oct4
promoter.
Claim 36
Independent Claim
17 words
A method according to any of Claims 33-35 wherein the selectable marker is the
neomycin phosphotransferase gene.
Claim 37
Dependent Claim
100 words
An animal cell capable of being cultured to form a mixture of cells including desired
stem cells and cells other than the desired stem cells, characterised in that all cells in the said
mixture of cells contain a selectable marker and in that in the said mixture of cells, under
appropriate selective culture conditions, differential expression of the selectable marker in (a)
the desired stem cells and (b) cells other than the desired stem cells enables selective survival
or growth of the desired stem cells to occur, so as to enable isolation and/or enrichment
and/or propagation of desired stem cells.
Claim 38
Dependent Claim
17 words
An animal cell as claimed in Claim 37 wherein the selectable marker is an antibiotic
resistance gene.
Claim 39
Dependent Claim
63 words
A vector for use in genetically modifying cells so as to be suitable for use in a method
according to any of Claims 1 to 36, said vector comprising a first genetic component
corresponding to said selectable marker and a second genetic component which in the
genetically modified animal cells directly or indirectly results in the said differential
expression of the selectable marker.
Claim 40
Dependent Claim
41 words
A vector as claimed in Claim 39 in the form of an expression vector in which said
second genetic component includes control sequences which are differentially activated in (a)
desired stem cells and (b) cells other than the desired stem cells.
Claim 41
Dependent Claim
15 words
A vector as claimed in Claim 40 wherein the control sequence is the Oct4 promoter.
Claim 42
Dependent Claim
14 words
A vector according to Claim 41 wherein the selectable marker is an antibiotic marker.
Claim 43
Dependent Claim
13 words
A vector according to Claim 42 wherein the antibiotic marker is neomycin
phosphotransferase.
Claim 44
Dependent Claim
35 words
A vector according to any of Claims 39-43 which when used in the genetic
modification of cells for use in the method of any of Claims 1 to 36 is not integrated into the
genome.
Claim 45
Dependent Claim
34 words
A vector according to any of Claims 39-43 in which said second genetic component
includes sequences which enable at least a portion of the first genetic component to be
specifically integrated into the genome.
Claim 46
Dependent Claim
30 words
A vector according to any of Claims 39-45 which additionally includes recognition
sequences, eg lox P or FRT sites, which allow subsequent excision of the integrated construct
via site-specific recombination.
Claim 47
Independent Claim
37 words
A method of preparing a transgenic animal comprising obtaining a desired stem cell
according to the method of any of claims 1-36, excising the selectable marker from the
desired stem cell and generating the transgenic animal therefrom.
Claim 48
Independent Claim
76 words
A method of preparing a transgenic animal, said animal comprising a selectable marker
capable of differential expression in (a) desired stem cells and (b) cells other than desired
stem cells, the method comprising: providing a blastocyst; providing animal cells according to any of Claims 37-38; introducing the animal cells into the blastocyst; transferring the blastocyst to a recipient; and allowing an embryo to develop to a chimaeric animal to enable germline transmission
of the selectable marker.
References Cited (Backward Citations)
No citation records indexed.
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