Re: Chimaeric animal patent?

말씀하신 유럽 특허에 대응하는 미국 특허 (US5166065)의 클레임

입니다. 아마도 유럽특허도 동일하거나 최소한 아주 유사한 특허청구범위??
특허되었을 겁니다.

청구항 1을 보면 시험관내의 포유동물 배(embryo) 배아 간세포를 분리하는

방법을 청구한 것이어서, 인간과 다른 포유동물의 잡종을 만들 수 있다는

내용을 특허청구한 것은 아닌 것 같습니다.

다만 명세서 가운데 그런 내용이 있을 수는 있겠지요.

아직 살펴보지는 않았는데, 클레임 뒤에 명세서 내용도 첨부했습니다.

검토해 주실꺼죠? 헤..

We claim:

1. A method for the isolation of embryonic stem (ES) cells from mamma
lian embryos in vitro which method comprises deriving and maintaining
said embryos in culture medium containing an effective amount of recom
binant leukaemia inhibitory factor (LIF) for a time and under conditio
ns sufficient for the development of said ES cells.

2. The method according to claim 1 wherein the culture medium is free
of feeder cells.

3. The method according to claim 1 wherein the culture medium is Eagl
e's medium or modifications thereof or equivalent thereto.

4. The method according to claim 1 wherein the LIF is recombinant hum
an or murine LIF.

5. The method according to claim 4 wherein LIF is added to the cultur
e medium at a concentration of from 10 to 1,000,000 units/ml.

6. The method according to claim 5 wherein the LIF is added to the cu
lture medium at a concentration of from 100 to 100,000 units/ml.

7. The method according to claim 6 wherein the LIF is added to the cu
lture medium at a concentration of from 500 to 10,000 units/ml.

8. A method according to claim 1 wherein the effective time is from 1
day to 20 weeks.

9. The method according to claim 8 wherein the effective time is from
1 to 8 weeks.

10. A method for maintaining mammalian embryonic stem (ES) cells in v
itro while retaining their pluripotential phenotype which process comp
rises culturing said cells in a culture medium containing an effective
amount of recombinant leukaemia inhibitory factor (LIF) under conditi
ons sufficient to maintain said cells.

11. The method according to claim 10 wherein the culture medium is fr
ee of feeder cells.

12. The method according to claim 10 wherein the culture medium compr
ises Eagle's medium or modifications thereof or equivalents thereto.

13. The method according to claim 10 wherein the LIF is recombinant m
urine or human LIF.

14. The method according to claim 13 wherein the recombinant LIF is a
dded to the culture medium at a concentration of from 10 to 1,000,000
units/ml.

15. The method according to claim 14 wherein the recombinant LIF is a
dded to the culture medium at a concentration of from 100 to 100,000 u
nits/ml.

16. The method according to claim 15 wherein LIF is added to the cult
ure medium at a concentration of from 500 to 10,000 units/ml.

여기서부터 명세서입니다.

This invention relates to the use of a previously discovered and char
acterised molecule, leukaemia inhibitory factor (LIF), in the isolatio
n and propagation of embryonic stem cells in vitro.

Embryonic stem (ES) cells, the pluripotent outgrowths of blastocysts,
can be cultured and manipulated in vitro and then returned to the emb
ryonic environment to contribute normally to all tissues including the
germline (for review see Robertson, E. J. (1986) Trends in Genetics 2
:9-13). Not only can ES cells propagated in vitro contribute efficient
ly to the formation of chimaeras, including germline chimaeras, but in
addition, these cells can be manipulated in vitro without losing thei
r capacity to generate germ-line chimaeras (Robertson, E. J. et. al. (
1986) Nature 323:445-447).

ES cells thus provide a route for the generation of transgenic animal
s such as transgenic mice, a route which has a number of important adv
antages compared with more conventional techniques, such as zygote inj
ection and viral infection (Wagner and Stewart (1986) in Experimental
Approaches to Embryonic Development. J. Rossant and A. Pedersen eds. C
ambridge: Cambridge University Press), for introducing new genetic mat
erial into such animals. First, the gene of interest can be introduced
and its integration and expression characterised in vitro. Secondly,
the effect of the introduced gene on the ES cell growth can be studied
in vitro. Thirdly, the characterised ES cells having a novel introduc
ed gene can be efficiently introduced into embryos by blastocyst injec
tion or embryo aggregation and the consequences of the introduced gene
on the development of the resulting transgenic chimaeras monitored du
ring pre- or post-natal life. Fourthly, the site in the ES cell genome
at which the introduced gene integrates can be manipulated, leaving t
he way open for gene targeting and gene replacement (Thomas, K. R. and
Capecci, M. R. (1987) Cell 51:503-512).

However, it is known that ES cells and certain EC (embryonal carcinom
a) cell lines will only retain the stem cell phenotype in vitro when c
ultured on a feeder layer of fibroblasts (such as murine STO cells, e.
g. Martin, G. R. and Evans, M. J. (1975) Proc. Natl. Acad. Sci. USA 72
:1441-1445) or when cultured in medium conditioned by certain cells (e
.g. Koopman, P. and Cotton, R. G. H. (1984) Exp. Cell Res. 154:233-242
; Smith, A. G. and Hooper, M. L. (1987) Devel.Biol. 121:1-91). In the
absence of feeder cells or conditioned medium, the ES cells spontaneou
sly differentiate into a wide variety of cell types, resembling those
found during embryogenesis and in the adult animal. The factors respon
sible for maintaining the pluripotency of ES cells have, however, rema
ined poorly characterised.

In work leading to the present invention, it has been found that LIF
has the capacity to substitute for, or be added to, feeder layers (or
conditioned medium) in supporting the maintenance of pluripotential ES
cells in vitro.

LIF is a protein that has previously been purified, cloned and produc
ed in large quantities in purified recombinant form from both Escheric
hia coli and yeast cells. (International Patent Application No. PCT/AU
88/00093, filed Mar. 31, 1988.) LIF has been defined as a factor, the
properties of which include:

1. it has the ability to suppress the proliferation of myeloid leukae
mic cells such as M1 cells, with associated differentiation of the leu
kaemic cells; and

2. it will compete with a molecule having the defined sequence of mur
ine LIF or human LIF (defined in International Patent Application No.
PCT/AU88/00093) for binding to specific cellular receptors on M1 cells
or murine or human macrophages. In addition to the biological propert
ies previously disclosed for murine and human LIF, LIF has now been fo
und to have the following properties:

(a) it allows the derivation and maintenance in the absence of feeder
cells of the pluripotential phenotype in vitro of ES cells.

(b) it allows the aforementioned ES cells, after passage in vitro in
the presence of LIF, to contribute to somatic and germline cell tissue
s of chimaeric animals such as mice when re-introduced into the embryo
nic environment;

(c) it demonstrates selective binding to high affinity receptors on m
urine ES (EKcs-1 (previously known as CS1) and D3) and EC (PCC3-3A and
F9) cells; and

(d) specific binding of @125 I-LIF to high affinity receptors is not
in competition with insulin, IGF-I, IGF-II, acidic and basic FGF, TGF.
beta., TNF.alpha., TNF.beta., NGF, PDGF, EGF, IL-1, IL-2, IL-4, GM-CSF
, G-CSF, Multi-CSF nor erythropoietin, but is in competition with muri
ne and human LIF.

Accordingly, a first aspect of the present invention relates to a met
hod for the isolation of embryonic stem (ES) cells from animal embryos
in vitro which method comprises deriving ES cells from said embryos i
n culture medium, said culture medium containing an effective amount o
f leukaemia inhibitory factor (LIF), for a time and under conditions s
ufficient for the development of said ES cells. The embryos used may b
e isolated from animals including, but not limited to, humans and a nu
mber of other animal species such as birds (e.g. chickens), mice, shee
p, pigs, cattle, goats and fish.

A second aspect of the present invention, contemplates a process for
maintaining animal embryonic stem (ES) cells in vitro while retaining
their pluripotential phenotype, which process comprises culturing said
cells in a culture medium containing an effective amount of leukaemia
inhibitory factor (LIF) under conditions sufficient to maintain said
cells. The ES cells in accordance with this aspect of the invention in
clude cells from humans, mice, birds (e.g. chickens), sheep, pigs, cat
tle, goats and fish.

The LIF used in the culture medium is preferably recombinant LIF prod
uced, by way of example, in accordance with the methods described in I
nternational Patent Application No. PCT/AU88/00093 In accordance with
the present invention, it has been found that recombinant LIF and in p
articular recombinant human and murine LIF are effective substitutes f
or, or additives to, feeder layers or conditioned medium in maintainin
g ES cells in vitro. For the purposes of the present description recom
binant LIF is produced in E. coli and yeast using the methods describe
d in International Patent Application No. PCT/AU88/00093, however, it
is within the scope of the present invention to include recombinant LI
F produced in other hosts including mammalian and insect cells and to
synthetic LIF.

In another aspect, the present invention extends to ES cells derived
from animal embryos by passage in a culture medium containing LIF, to
such ES cells having additional genetic material inserted therein, and
to chimaeric animals such as chimaeric mice or transgenic progeny of
said animals generated by known techniques using ES cells which have b
een maintained in vitro in a LIF-containing culture medium.

Thus, the invention extends to the generation and maintenance of ES c
ells from humans, mice, birds (e.g. chickens), sheep, pigs, cattle, go
ats and fish and to the generation of transgenic chimaeric animals and
their transgenic progeny using the ES cells isolated from animal spec
ies such as mice, birds (e.g. chickens), sheep, pigs, cattle, goats an
d fish. This invention also includes the use of LIF in culture media t
o modulate the survival and growth of human and other animal species s
uch as cattle germ cells and embryonic cells, for example, for use in
in vitro fertilisation and other procedures.

The present invention may also be described by reference to the follo
wing figures:

FIGS. 1A and 1B are graphical representations showing the effect on E
S cells of different concentrations of LIF.

FIGS. 2A, 2B, 2C, 2D, 2E and 2F are representations showing ES cell m
orphology in the presence and absence of LIF.

FIG. 3 is a graphical (3A and 3C) and pictorial (3B) representation s
howing the binding of @125 I-LIF to ES cells (EKcs-1) and EC cells (F9
and PCC3-A).

The present invention is directed to a method for the isolation and m
aintenance of embryonic stem (ES) cells from animal embryos in vitro w
hich method comprises deriving and/or maintaining said ES cells from s
aid embryos in culture medium containing an effective amount of leukae
mia inhibitory factor (LIF), for a time and under conditions sufficien
t for the derivation and/or maintenance of said ES cells. The animal e
mbryos may be isolated from a number of animal species such as humans,
mice, birds (e.g. chickens), sheep, pigs, cattle, goats and fish. By
reference herein to "animal embryos" includes reference to "animal bla
stocyts. Furthermore, the present invention is exemplified using human
LIF with murine ES cells (heterologous system) and murine LIF with mu
rine ES cells (homologous system) This is done with the understanding
that the present invention contemplates LIF from any animal species in
heterologous or homologous systems with animal embryos from animal sp
ecies such as humans, mice, birds (e.g. chickens), sheep, pigs, cattle
, goats and fish. Although in certain circumstances, a heterologous sy
stem will work effectively, it may be preferable to use homologous sys
tems. Given the teachings herein, it will be routine for the skilled t
echnician to ascertain whether a homologous or heterologous system is
required in order to isolate or maintain particular animal ES cells.

By "culture medium" is meant a suitable medium capable of supporting
growth of ES cells. Examples of suitable culture media useful in pract
icing the present invention are Eagles medium or modifications or equi
valents thereof such as Dulbecco's or Glasgows modified Eagle's medium
with supplements such as 5%-30% (v/v) foetal calf serum and where nec
essary 0.01 to 1.0 mM .beta.-mercaptoethanol but preferably about 0.1
mm .beta.-mercaptoethanol. The culture medium may or may not contain f
eeder cells and LIF may be used to substitute for, or add to, said fee
der cells. When required, LIF, or more particularly synthetic or recom
binant LIF, is added to the medium at a concentration of about 100-1,0
00,000 units/ml and preferably about 100-100,000 units/ml and even mor
e preferably 500-10,000 units/ml where 50 units are defined as the amo
unt of LIF which in one milliliter induces a 50% reduction in clone fo
rmation by murine M1 myeloid cells. By "recombinant LIF" is meant the
LIF prepared by genetic engineering means such as, for example, accord
ing to International Patent Application No. PCT/AU88/00093 where a num
ber of hosts such as bacteria (e.g. E. coli) or yeast cells may be emp
loyed. In accordance with the present invention, the effective derivat
ion time is from 1 day to 20 weeks and particularly from 1 to 8 weeks.

Another aspect of the present invention contemplates a process for ma
intaining animal ES cells in vitro while retaining their pluripotentia
l phenotype which process comprises culturing said cells in a culture
medium containing an effective amount of LIF under conditions sufficie
nt to maintain said cells. The ES cells in accordance with this aspect
of the invention include cells derived from humans, mice, birds (e.g.
chickens), sheep, pigs, cattle, goats and fish. As with the isolation
of ES cells from animal embryos, the LIF used in the aformentioned pr
ocess is preferably recombinant LIF. The culture medium may or may not
contain feeder cells.

In accordance with the present invention, "pluripotential cells" and
"embryonic stem cells" are those which retain the developmental potent
ial to differentiate into all somatic and germ cell lineages.

The ability of recombinant LIF to maintain the stem cell phenotype of
ES cells is demonstrated by transferring ES cells D3 and HD5 into nor
mal cell culture medium in the presence of varying concentrations of p
urified yeast-derived recombinant human LIF (rY-HLIF) or E. coli--deri
ved recombinant mouse LIF (rE-MLIF). At concentrations of 1000-5000 un
its/ml of rY-HLIF or rE-MLIF more than 90% of the D3 and HD5 ES cells
retained their stem cell phenotype. In contrast, the ES cells maintain
ed in normal culture medium differentiated over a period of 3-6 days.
The proportion of colonies having the stem cell phenotype was related
to the concentration of LIF in the culture medium. In addition to main
taining established ES cell lines, six new ES cell lines (MBL-1,2,3,4,
5 & 6) were isolated from blastocysts in the absence of feeder cells w
hen the media was supplemented with 1000 units/ml rE-HLIF Long term ma
intenance of the ES cell lines D3, HD5 and MBL-1 to 6 in LIF for up to
22 passages (approximately 100 cell generations or 10 weeks) did not
noticeably alter the growth characteristics of these ES cells or their
dose dependency on LIF. The ability of these ES cells to differentiat
e into all somatic and germ cell linages was confirmed by reintroducti
on of D3 and MBL-1 cells into blastocysts. Approximately 50% of the pr
ogeny analysed contained tissues derived from the injected ES cells wi
th levels of overt chimaerism as high as 90% in individual mice. To te
st for germline transmission of ES derived cells male chimaeras were m
ated to C57BL/6J mice. Three D3 and two MBL-1 C57BL/6J chimaeras gave
rise to agouti progeny confirming that these ES cells can contribute t
o the formation of germ cells.

The present invention also relates to chimaeric animals generated by
known techniques using the ES cells contemplated herein. These ES cell
s may be isolated from animal embryos and/or maintained in vitro accor
ding to the subject invention. Furthermore, genetically manipulated ES
cells may be passaged in LIF and used to make chimaeric animals. For
example, genetically manipulated ES cells containing a retrovirus vect
or (N-TK527; derived from pXTl; C. A. Boulter and E. F. Wagner, (1987)
Nucl. Acids Res. 15:7194) encoding genes for neomycin resistance and
c-src@527 were propogated in the presence of LIF but in the absence of
feeder cells for over 20 passages. These cells still retained the abi
lity to differentiate as judged by the formation of normal chimaeras f
ollowing introduction of these cells into preimplantation embryos by b
lastocyst injection.

Further details of the use of LIF in accordance with the present inve
ntion will be apparent from the following Examples.

EXAMPLE 1

This example sets out the steps used to maintain ES cells in vitro in
LIF, and to generate chimaeric mice using ES cells so passaged.

Step 1: Propagation in vitro

The ES cells used were the D3 (Doetschman, T. C. et. al. (1985) J.Emb
ryol.Exp.Morphol. 87: 27-45) the EKcs-1 (Previously known as CS1) (Wag
ner, E. F. et.al. (1985) Cold Spring Harbor Symp.Quant.Biol. 50:691-70
0) and the HD5 (C. Stewart, unpublished) ES cell lines isolated from 1
29 SV He blastocysts and the CBL63 (R. Kemler, unpublished) ES cells i
solated from C57BL/6J blastocysts. Prior to culture in LIF, the D3 and
CBL63 cells were maintained in Dulbecco modified Eagles medium with 1
5% (v/v) foetal calf serum on a feeder layer of primary embryo fibrobl
asts, and the EKcs-1 and HD5 ES cells were maintained in Eagle's mediu
m with 15% (v/v) foetal calf serumand 0.1 mM .beta.-mecraptcethanol, i
n the presence of medium conditioned by the bladder carcinoma cell lin
e 5637 (ATCC No.HTB9).

The ability of recombinant LIF to maintain the stem cell phenotype of
ES cells was demonstrated by transferring ES cells of the lines D3 an
d HD5 into normal cell culture medium in the presence of varying conce
ntrations of purified yeast-derived recombinant human LIF (hereafter r
eferred to as rY-HLIF), or E.coli derived recombinant mouse LIF (rE-ML
IF) (previously disclosed in International Patent Application No. PCT/
AU88/00093). The results are shown in FIGS. 1 and 2. In FIG. 1A, HDS c
ells previously maintained in 80% 5637 conditioned medium for eight pa
ssages were transferred to culture media containing 0-5,000 units m1@-
1 of purified, recombinant yeast-derived human LIF (H-LIF; see below)
( ) or purified, recombinant E. coli-derived mouse LIF (M-LIF; see bel
ow) (o--o). HD5 cells maintained in medium containing 1,000 units ml@-
1 H-LIF for a further 13 passages were then transferred to 0-1,000 uni
ts ml@-1 M-LIF (). In FIG. 1B, D3 cells maintined on mouse embryo fibr
oblasts for 10 passages were transferred to media containing 1,000-5,0
00 units ml@-1 H-LIF and after a further 7 or 15 passages the cells we
re transferred into media containing 0-5,000 units ml@-1 of H-LIF () o
r 0-1,000 units m@-1 M-LIF () respectively. FIG. 2 shows ES cell morph
ology in the presence of recombinant LIF. HD5 ES cells cultured in the
presence of 80% 5637 conditioned medium were assayed for the ability
of purified recombinant LIF to maintain the stem-cell phenotype by tra
nsfer to media containing 1,000 units ml@-1 M-LIF (A), or to normal cu
lture media (B). After seven days, the colonies were srained with Giem
sa. Compact stem-cell colonies could be distinguished from diffuse dif
ferentiated colonies. D3 cells maintained in H-LIF for 15 passages wer
e assayed for the ability to differentiate by transfer into media cont
aining 1,000 units ml@-1 M-LIF (C) or normal culture media (D). Immuno
fluorescence of the cells in the two D3 colony types was carried out u
sing the ECMA-7 monoclonal antibody which recognizes a stem cell-speci
fic cell-surface antigen. Cell-surface-specific immunofluorescence was
detected on over 90% of the cells maintained in media containing 1,00
0 units ml@-1 recombinant LIF (E) but on less than 1% of the cells mai
ntained in normal culture media (F). The field of view shown in (F) co
ntains 21 cells.

FIGS. 1 and 2 indicate that over 90% of the ES cells maintained in 10
00-5000 units/ml rY-HLIF or rE-MLIF retained their stem cell phenotype
. In contrast, ES cells maintained in normal culture medium differenti
ated over a period of 3-6 days. The different concentrations of rY-HLI
F or rE-MLIF used did not result in any noticeable change in cell numb
er after 6 days in culture, indicating that there is no selection for
a specific subpopulation able to grow in LIF. Similar results have bee
n obtained using yeast-derived rMLIF also disclosed in International P
atent Application No. PCT/AU88/00093. The data in FIG. I indicate that
human LIF acts on mouse ES cells, as previously described for the act
ion of human LIF on Ml myeloid leukaemic cells (Gough, N. M. et. al. (
1988) Proc.Natl.Acad.Sci.USA 85: 2623-2627). The data in FIG. I also i
ndicate that the action of LIF on ES cells is independent of glycosyla
tion, as previously described for the action of LIF on M1 myeloid leuk
aemic cells.

Four ES cell lines, D3, EKcs-1, CBL63 and HD5, were maintained in med
ium containing 1000-5000 u/ml rY-HLIF for up to 22 passages (10 weeks
or approximately 100 generations). Long-term maintenance of the ES cel
ls in rY-HLIF did not noticeably alter the growth characteristics of t
he cells. Furthermore, reduction or removal of the LIF from the cultur
e medium resulted in the differentiation of the ES cells with similar
kinetics to those explanted directly from bladder carcinoma 5637 condi
tioned medium or a feeder layer of mouse fibroblasts (for example, see
FIGS. 1 and 2). The stem cell phenotype of ES cells cultured for mult
iple passages in the presence of LIF was confirmed by immunofluorescen
ce with the ECMA-7 antibody which recognises a cell-surface stem-cell-
specific antigen (Kemler, R. in Progress in Developmental Biology Band
26 Sauer, H. W. ed page 175; Fisher, Stuttgart, 1980); ES cells cultu
red in the presence of LIF expressed the stem cell marker, whereas in
the absence of LIF less than 1% did so (FIG. 2).

Step 2: Isolation of ES cell lines

Murine blastocysts were isolated from 129 Sv He mice at day 4 of deve
lopment (day 1=day of plug) into either Dulbecco's or Glasgows modifie
d Eagle's medium with 15% (v/v) foetal calf serum, 0.1 mM .beta.-merca
ptoethanol and 1000 units/ml of purified rE-HLIF. ES cell lines were t
hen isolated by two different methodologies.

In the first method the blastocysts were allowed to attach to the cul
ture dish and approximately 7 days later the outgrowing inner cell mas
s picked, trypsinised and transfered to another culture dish in the sa
me culture media. ES cell colonies appeared 2-3 weeks later with betwe
en 5-7 individual colonies arising from each explanted inner cell mass
. The ES cell lines were then expanded for further analysis. The secon
d method for isolation of ES cell lines used the immunosurgery techniq
ue (described in Martin, G. R. (1981) Proc. Natl. Acad. Sci. USA 78:76
34-7638) where the trophectoderm cells are destroyed using anti-mouse
antibodies prior to explanting the inner cell mass. The efficiency of
ES cell lines isolation is shown in Table 1.

Step 3: Generation of Chimaeric Mice

All the ES cell lines cultured in the absence of feeder cells but in
the presence of LIF (referred to in step 1) or directly isolated with
the aid of culture medium containing LIF (referred to in step 2) retai
ned the ability to differentiate into multiple cell types following th
e removal of LIF indicating that these cells have retained their pluri
potential phenotype. To confirm their developmental potential, D3 ES c
ells maintained in LIF for 7-22 passages and MBL-1 ES cells maintained
in LIF for 14-17 passages were reintroduced into the embryonic enviro
nment by blastocyst injection (as described in Williams et al., (1988)
Cell 52:121-131). Blastocysts were isolated from the outbred ICR mous
e strain or inbred C57BL/6J mice. The expanded blastocysts were mainta
ined in oil-drop cultures at 4 DEG C. for 10 min prior to culture. The
ES cells were prepared by picking individual colonies, which were the
n incubated in phosphate-buffered saline, 0.5 mM EGTA for 5 min; a sin
gle cell suspension was prepared by incubation in a trypsin-EDTA solut
ion containing 1% (v/v) chick serum for a further 5 min at 4 DEG C. Fi
ve to twenty ES cells (in Dulbecco's modified Eagle's Medium with 10%
(v/v) foetal calf serum and 3,000 units/ml DNAase 1 buffered in 20 mM
HEPES [pH 8]) were injected into each blastocyst. Blastocysts were tra
nsferred into pseudopre.gnant recipients and allowed to develop normal
ly. Chimaeric mice were identified by coat markers (Hogan et al., (198
6) Manipulating the Mouse Embryo, Cold Spring Harbor, N.Y.). Analysis
of the subsequent chimaeric mice revealed that up to approximately 50%
of the progeny contained tissues derived from the injected cells (Tab
le 2), with levels of overt chimaerism as high as 90% in individual mi
ce. Furthermore analysis of the organs of four D3-chimaeras confirmed
that the ES cells maintained in LIF could contribute extensively to th
e development of all of the somatic tissues (Table 3).

The male chimaeras were tested for germline transmission of ES derive
d cells by mating to ICR or C57BL/6J females. Three out of four of the
D3-C57BL/6J chimaeras and two out of six of the MBL-1-C57BL/6J chimae
ras gave rise to agouti offspring derived from the ES cells cultured i
n LIF (Table 4).

To test whether genetically altered ES cells could be maintained in c
ulture medium containing LIF, D3 ES cells were infected with a retrovi
rus vector (N-TK527) expressing the neomycin resistance gene and a C-s
rc gene mutant (c-src@527) (protocol for infection is described in Wil
liams et al., (1988) Cell 52: 121-131). The ES cell clones isolated we
re maintained in culture medium containing LIF for over 20 passages. T
hese genetically modified ES cells retained the ability to form chimae
ric mice following reintroduction into the embryonic environment by bl
astocyst injection (Table 2)

TABLE 1

______________________________________

Isolation of 129 Sv He ES cell lines in media

containing rE-HLIF

ICM Number of ES cell

Methodology

Blastocyst

outgrowing

lines derived

______________________________________

Explanted 9 9 4

Immunosurgery

11 3 0

Immunosurgery

7 5 2

______________________________________

Murine blastocysts were isolated from 129 Sv He mice at day 4 of deve
lopment (day 1=day of plug) into either Dulbecco's or Glasgows modifie
d Eagle's medium with 15% (v/v) foetal calf serum, 0.1 mM .beta.-merca
ptoethanol and 1000 units/ml of purified rE-HLIF. The blastocysts were
then explanted into the same media and left to attach to the culture
dish and the inner cell mass picked dissociated in phosphate-buffered
saline, 0.5 mM EGTA for 5 min; a single cell suspension was prepared b
y incubation in a trypsin-EDTA solution containing 1% (v/v) chick seru
m and the cells replated in the cell culture medium described above. T
he characteristic ES cell colonies appeared within 1-3 weeks.

Other blastocysts were treated by immunosurgery (as described in Mart
in, G. R. (1981) Proc. Natl. Acad. Sci. USA 78:7634-7638). The blastoc
ysts were allowed to hatch from the zona pelucida, and then treated wi
th anti-mouse antibodies and destroyed by the addition of complement.
The exposed inner cell mass was then left to attach to a tissue cultur
e dish and again treated with anti-mouse antibodies and complement. Wi
thin a few days pluripotential stem cell colonies appeared and were di
ssociated and trypsinised as described above.

TABLE 2

______________________________________

Chimaeric mice derived from ES cells cultured in LIF

ES Blastocysts Pups

cells transferred born Chimaeras

______________________________________

D3 142 60 (42%) 33 (55%)

MBL-1 51 33 (65%) 16 (48%)

D3 N-TK527 42 22 (52%) 12 (54%)

______________________________________

TABLE 3

______________________________________

Percentage tissue contributions in individual D3 chimaeric mice

Chimaera

Necropsy age

______________________________________

C Bl Sp P Li T H

D3-1 13d 35 0 35 20 10 20 40

D3-2 14d 40 15 35 30 45 30 50

D3-3 11d 90 50 50 35 50 40 60

D3-4 11d 50 50 50 30 40 40 50

Lu G K M B Sa

D3-1 13d 30 10 35 30 35 20

D3-2 14d 35 20 30 50 50 25

D3-3 11d 45 50 50 70 50 55

D3-4 11d 50 35 50 50 20 30

______________________________________

TABLE 4

______________________________________

Chimaeric demonstrating germline transmission of ES

derived cells.

Passage no. of D3 cells

Offspring

Mice Chimaerism

on feeders in LIF 129 Sv He

C57

______________________________________

775-3

75% 10 16 9 24

778-1

70% 10 22 5 33

778-2

50% 10 22 2 36

778-3

55% 10 22 0 0

______________________________________

The following relates to Tables 2, 3 and 4:

D3 and MB1-1 ES cells are derived from 129 Sv He mice (inbred, agouti
, homozygous for the glucose phosphate isomerase 1@a allele). The D3 E
S cells were originally cultured on primary embryo fibroblasts for 10
passages and then transferred to 1,000-5,000 units/ml recombinant LIF
for 7-22 passages. The MB1-1 ES cells were isolated in the absence of
feeder cells but in the presence of rE-HLIF these cells were cultured
for 14-17 passages. The ES cells were then injected into ICR (outbred,
albino) or C57BL/6J (inbred, black) blastocysts which were then trans
ferred into pseudo-pre.gnant foster mothers. Both the ICR and C57BL/6J
mice are homozygous for the glucose phosphate isomerase 1@b allele. C
himaeric pups were identified by coat pigmentation (only foster mother
s which became pre.gnant were counted in estimating the number of prog
eny). Tissue chimaerism was estimated using glucose phosphate isomeras
e strain differences. The extent of tissue chimaerism was determined i
n two D3-ICR (numbers 1 and 2) and two D3-C57BL/6J chimaeras (numbers
3 and 4). Tissues analysed: C, coat; Bl, blood; Sp, spleen; P, pancrea
se; Li, liver; T, thymus; H, heart; Lu, lungs; G, gonads; K, kidneys;
M, muscle; B, brain; Sa, salivary gland. Male chimaeras were mated to
ICR or C57BL/6J mice and offspring identified by coat pigmentation.

EXAMPLE 2

This example sets out the steps used to document specific high affini
ty receptors on ES and EC cells. Accompanying FIG. 3 shows binding of
@125 I-LIF to ES cells EKcs-1 and EC cells F9 and PCC3-A (Jakob, J. et
. al. (1973) Ann.Microbiol.Inst.Pasteur, 124B: 269-282). In relation t
o FIG. 3, (A), Scatchard analysis of @125 I-labelled LIF binding to F9
( ), EKcs-1 (), PCC3A-1 () and M1 (.smallcircle.) cells. Saturation c
urves for binding were analysed by the method of Scatchard by plotting
the amount of LIF specifically bound (defined as the difference betwe
en binding observed in the absence and presence of excess unlabelled L
IF) versus the ratio of bound to free LIF. Free LIF values were adjust
ed for the percent of @125 I-labelled LIF capable of binding specifica
lly to LIF receptors, in this experiment determined to be 75%. The app
arent dissociation constant for the interaction of LIF with its recept
or was determined from the slopes of the curves and the receptor numbe
r from their intercepts with the ordinate. Results in (A) were standar
dized to 5.times.10@6 cells per point and the mean of duplicate points
are shown and curves were fitted using the Ligand program (B), Autora
diography of F9 EC cells labelled with @125 I-labelled LIF. (C), Quant
itation of silver grains on F9 EC cells after binding of @125 I-labell
ed LIF.

Purified recombinant (yeast-derived) human LIF (rY-HLIF) was radioact
ively labelled on tyrosine residues as described previously (Hilton, D
. J. et.al. (1988) Proc.Natl.Acad.Sci. USA, 85:5971-5975) producing @1
25 I-LIF with a specific radioactivity of approximately 1.2.times.10@7
cpm/pmole. @125 I-LIF (2.times.10@3 -5.times.10@5 cpm) was incubated
with 1-4.times.10@6 target cells with or without at least 100-fold mol
ar excess of unlabelled LIF, in a total volume of 100 .mu.l for 4 hour
s on ice. Cell-associated and free @125 I-LIF were separated by centri
fugation through foetal claf serum (Nicola, N. A. and Metcalf, (1986)
D. J.Cell Physiol. 128:160-188). Specific cell-associated @125 I-LIF w
as determined by cold competition.

FIG. 3 illustrates the specific saturable and high affinity binding o
f @125 I-LIF to the ES cells EKcs-1 and the EC cells PCC3-A and F9. Th
e number of LIF receptors per cell derived from these Scatchard plots
were 295, 190 and 330, respectively, with apparent dissociation consta
nts at 4 DEG C. of approximately 90 pM. This compares with the M1 cell
line, a LIF-responsive monocytic leukaemia, which displays 50-200 LIF
receptors/cell with an apparent dissociation constant of 50-150 pM. A
ll other ES and EC cells tested - D3, NG2, PC13 and P19 - bound simila
r levels of LIF (data not shown).

The binding of @125 I-LIF to M1 cells, EKcs-1 and PCC3-A was also fou
nd to be in competition with unlabelled recombinant and native murine
and human LIF, but not with the range of other hormones and factors, (
including several which act on embryonic cells): insulin, IGF-I, IGF-I
I, acidic and basic FGF, TGF.beta., TNF.alpha., TNF.beta., NGF, PDGR,
EGF, IL-1, IL-4, GM-CSF, G-CSF, Multi-CSF and erythropoietin.
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