Note: Descriptions are shown in the official language in which they were submitted.
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WO97/41209 PCT/EP97/02323
PLURIPOTENT RABBIT EMBRYONIC STEM (ES) CELL LINES AND
THEIR USE IN THE r,~N~TION OF CHIMERIC RABBITS
The present invention relates to novel rabblt
embryonic stem (ES) cell lines and their use in the gene-
ration of chimerlc rabbits.
Gene targeting (via homologous recombination in
5 embryonic stem (ES) cells) technology allows the
manipulation of the genome in desired and defined ways
(Capecchi, 1989; Robertson, 1987; Bradley, 1987).
Briefly, genes to be targeted are incorporated in plasmid
transfer vectors and altered by replacement of some of
10 their sequence with foreign DNA which encodes selectable
markers (inactivation) or by a mutated gene sequence
(targeted mutagenesis). These inactivated or mutated
genes are then introduced into ES cells (which each have
the potential to develop into a complete animal).
15 Subsequently clones of ES cells in which the natural gene
is replaced by the inactivated hybrid are selected ln
vitro, and these selected clones are introduced into
normal embryos which are reimplanted. This process
generates chimeric animals which are selected for
20 germline transmission of the inactivated gene. Through
breeding and selection, transgenic animals are then gene-
rated which are deficient in the targeted ("knocked-out")
gene.
Completely ES cell derived mice can be
25 generated utilising the recently developed technology to
aggregate wild type or mutant ES cells with tetraploid
embryos (Nagy et al., 1993). Alternatively ES cells can
be used for introduction of genetic material by non-
homologous recombination, allowing the study of genetic
30 alteration in live animals.
The application of transgenic technologies and
ES cell technologies for the alteration of gene function
- has provided animal models of important human diseases
(for review cfr. Wilson, 1996; Rubin and Barsch, 1996).
35 However, at present this technology has only been
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WO97/41209 PCT~P97/02323
successful in the mouse. Although useful for many
applications, the mouse has significant restrictions
(e.g. size and inability to generate the phenotype of
human diseases) that limit its potential applications.
5 Consequently, larger animal models to test the phenotypic
consequences or loss-of-function mutations would be very
valuable.
Presumptive pluripotential ES cells have been
isolated in a number of additional species including
10 hamster (Doetschman et al., 1988), pig tEvans et al.,
1990; Piedrahita et al., 1990; Notarianni et al., 1990;
Talbot et al., 1993), sheep (Notarianni et al., 1990),
cattle (Evans et al.; Saito et al., 1992), mink (Sukoyan
et al., 1993), rabbit (Graves and Moreadith, 1993), rat
15 (Iannaccione et al., 1994), man (Bongso et al., 1994) and
primate (Thomson et al., 1995). However, only in the
mouse and rat ES cells have been established in culture
giving rise to chimeras following reintroduction into
blastocysts, whereas all efforts in the rabbit and other
20 species have failed to date.
Inner cell mass (ICM) cells freshly derived
from rabbit blastocysts have been shown to allow the
generation of chimeric rabbits following injection into
recipient blastocysts (Gardner and Munro, 1974; Moustafa,
25 1974; Babinet and Bordenave, 1980), whereas Yang et al.
(1993) have reported the production of chimeric rabbits
both from freshly isolated ICM and from ICM cells
maintained in culture in vitro for 3 days.
However, in order to allow gene targeting by
30 homologous recombination in ES cells, cell lines that can
be kept in culture for a longer period of time and with
demonstrated potential to generate chimeric animals are
necessary for the production of offspring with targeted
genetic alterations. Prior to the present invention, no
35 such cell lines of animals other than mice and rats had
- been isolated.
The derivation of putative pluripotential
rabbit ES cells was previously reported by Graves and
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WO97/41209 PCT~P97/02323
Moreadith (1993). Two principal cell types emerged follo-
wing serial passage of exp~anted embryos. One type had
morphology identical to pr~mary outgrowths of trophecto-
derm, cells that are responsible for embryo implantation.
5 The second type appeared t~pically epitheloid and was
presumed to represent rabbit ES cells (Figure 1).
Although these putative ES cells were able to generate
embryoid bodies, consisting of multiple cell types
representative of ectoderm, mesoderm and endoderm, they
10 were unable to produce chi~eric rabbits after
introduction of the epitheloid cells into blastocysts
from the New Zealand White (NZW) strain (Graves and
Moreadith, 1993; Graves and Moreadith, unpublished
results (Table 1)) or after nuclear transfer into a
15 denucleated New Zealand White zygote (~u et al., 1995).
It was suggested that the inability to produce chimeras
with these ES cells could have been due to a strain
barrier. Nevertheless, the data in Table l demonstrate
that no chimeric animals could be derived from any of the
20 four tested epitheloid lines, each derived from a single
inner cell mass.
Table l: Results of rabbit embryo injection with putative
ES cell lines (unpublished results, Graves and
Moreadith)
cell line stage of #injections #born (~) chimeras
(%)
embryo~0
-
GM3 blastocyst 46 29(63) 0(0)
GM4 blastocyst 24 2 (8) 0(0)
GM8 morula 84 6 (7) 0(0)
35 GM4 morula 14 5(35) 0(0)
Total 168 42(25) 0(0)
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WO97/41209 PCT~P97/02323
Niemann and Strelchenko (1994) also attempted
to isolate and maintain pluripotent rabbit (California
strain) ES cells but, although aggregation of the
putative ES cells (maintained for a total of 12 passages)
5 with the inner cell mass of the host blastocyst
(Chinchilla, Black Rex) was observed, generation of
chimeric offspring was not documented. These putative ES
cells were derived from rabbit embryos collected 4 days
after mating. Thus, to date, all efforts to generate
10 stable pluripotent rabbit ES cell lines, with the
demonstrated potential to generate chimeric animals
following injection into recipient blastocysts, have
failed.
It is therefore the object of the invention to
15 come to the improved derivation and maintenance of rabbit
embryonic stem (ES) cells with demonstrated potential to
generate chimeric rabbits following injection in
recipient blastocysts, thus enabling the production of
wild type or genetically altered offspring (via either
20 homologous or non-homologous recombination, using for
example gene or nuclear transfer).
It was now found that a rabbit embryonic stem
(ES) cell line, comprising at least 70~, preferably 80 to
9o~ undifferentiated cells is obtainable by isolating the
25 inner cell mass of 5.5 days postcoitus blastocysts and
culturing them on feeder cells in rabbit ES medium.
The rabbit ES medium according to the invention
comprises high glucose Dulbecco's Modified Eagle Medium,
4mM L-glutamine, 0.1 mM 2-mercaptoethanol, 148 units/ml
30 penicillin G sodium, 148 microgram/ml streptomycin
sulfate, 4 microgram/ml bovine insulin, 103 units/ml
murine Leukemia Inhibitory Factor, 20~ fetal bovine
serum, 1.5~ MEM non-essential amino acid solution.
The feeder cells are preferably mouse embryonic
35 fibroblasts derived from 12.5 days old mouse embryos and
- used in a density of 3 to 4X106 cells per 10 cm petri
dish.
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PCT~P97/02323
WO97/41209
Preferably also a modified trypsinization
method was used, which consists of the use of
trypsinization medium comprising O.l~ collagenase, l~
chicken serum and 0.03~ trypsin-EDTA in phosphate-
5 buffered saline. This medium allowed the selectivepassage of ES cells because mouse embryonic fibroblasts
and trophectodermal cells detach more slowly in this
medium.
Cells according to the invention are
lO recognisable by virtue of their following properties:
three dimensional colony formation, positive staining for
alkaline phosphatase and negative staining for
cytokeratin 18 and vimentin after more than lO passages.
The invention further relates to the use of the
15 ES cell lines for the generation of chimeric rabbits, for
example following blastocyst injection into recipient
blastocysts or embryo aggregation or nuclear transfer.
The ES cell lines of the invention may also be
used for gene alteration by homologous or non-homologous
20 recombination or for the generation of rabbits with gene
alteration via germline transmission.
The use or differentiation of cell lines accor-
ding to the invention can lead to the study or isolation
of (novel) genes.
The present invention thus leads to the
improved derivation of pluripotent ES cells, their
maintenance in culture over several passages, and their
use for the successful generation of chimeric animals.
The derivation and maintenance of rabbit ES
cells with demonstrated potential to generate chimeric
rabbits following injection in recipient blastocysts will
allow the generation of offspring capable of germline
transmission of the targeted mutations following
homologous or non-homologous recombination in these
pluripotent ES cells. In addition, the pluripotency of
the rabbit ES cells will allow to differentiate them into
defined cell types enabling the study or isolation of
novel genes.
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WO97/41209 PCT~P97/02323
The invention will be illustrated in the
following examples, that are not intended to limit the
scope of the invention. Based on the present invention,
several variants and improvements will be obvlous to
those skilled in the art.
_ _
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WO97/41209 PCT~P97/02323
EXAMP~E 1
Cell culture conditions
Starting from the ES cell line GM3, derived
from Dutch Belted rabbit embryos as described by Graves
and Moreadith (1993) (Figure 1), cell culture conditions
were developed which stabilized the percentage of
undifferen-tiated ES cells, relative to the methods
described by Graves and Moreadith (1993). Thus, it became
possible, for the first time, to generate chimeras after
injection of ES cells into the blastocoel cavity of New
Zealand White blastocysts, as will be demonstrated in
Example 2.
Changes were made to the cell culture medium,
the density of the mouse embryonic fibroblast (MEF)
feeder layers, the age of the mouse embryos used to
derive the MEFs, and the trypsinization medium used to
dislodge cells for passage.
The culture medium used by Graves and Moreadith
(1993) consisted of high glucose Dulbecco's Modified
Eagle Medium, 4mM L-glutamine, 0.1 mM 2-mercaptoethanol,
penicillin G sodium 148U/ml, and streptomycin sulfate 148
~g/ml.
According to the invention the following
supplements were changed or added: 4 ~g/ml bovine
insulin, 103 units/ml of murine Leukemia Inhibitory
Factor, 20~ fetal bovine serum, 1.5~ MEM non-essential
amino acid solution.
The rabbit ES cells (1.5 to 3 X 106 cells per 10
cm petridish) were grown to subconfluency on mouse
embryonic fibroblasts mitotically arrested with mitomycin
and the ES cells were passaged every 4-6 days onto
freshly prepared feeders (3 to 4 X 106 cells per 10 cm
dish). The ES cells were fed every day with the improved
medium described above. Culture dishes were kept at 39~C
in a humidified atmosphere of 5~ C02 in air. The mouse
embryonic fibroblast were derived from 12.5 day old mouse
embryos and were used at passage 1. The increased density
of the mouse embryonic fibroblasts (3 to 4 X lo6 as
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WO97/41209 PCT~P97/02323
compared to 2 to 3 X 106 cells per 10 cm dish (Graves and
Moreadith, 1993)) together with the use of 12.5 days
embryos markedly reduce-d the differentiation of the ES
cells.
This reduction of differentiation proved to be
crucial, since only undifferentiated cells maintain the
capacity to incorporate in the inner cell mass of a
recipient embryo, a prerequisite to give rise to chimeric
offspring.
Furthermore, an improved selective
trypsinization method was used which allowed the removal
of trophectodermal cells (which can induce ES cell
differentiation) from the culture. The trypsinization
medium consisted of 0.1~ collagenase, 1~ chicken serum
and 0.03~ trypsin-EDTA (Gibco Cat. no. 25200) in
phosphate buffered saline. This selective trypsinization
medium allowed the selective passage of ES cells because
mouse embryonic fibroblasts and trophectodermal cells
detach more slowly from the culture dish than ES cells.
EXAMPLE 2
Generation of chimeras
The GM3 cells, derived from Dutch Belted
embryos by Graves and Moreadith (1993), but maintained in
culture conditions in Example 1, were used to generate
chimeric offspring as described below. Blastocyst
injection of the epitheloid colonies of the putative ES
cells had previously never generated chimeric rabbits
after injection into rabbit embryos (Table 1). For
further experiments described below, GM3 cells from
passage 12 were maintained in improved culture conditions
which stabilized the percentage of alkaline phosphatase
positive, undifferentiated ES cells.
Sexually mature New Zealand White females were
superovulated with six consecutive subcutaneous
injections of porcine follicle stimulating hormone (FSH -
0.4, 0.4, 0.5, 0.5, 0.5, 0.5 mg) given 12 hours apart,
followed by 75 IU of human chorionic gonadoptropin (hCG)
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WO97/41209 PCT~P97/02323
given intravenously 10 hours after the last dose of FSH.
Following the hCG injection, the females were mated with
mature males of the same strain.
Blastocysts were recovered from the uterine
horns 90 hours after mating, by flushing the uterine
cavity with Dulbecco's phosphate buffered saline
supplemented with 3~ bovine serum albumin (Cohn fraction
V) plus 5~ antibiotic/antimycotic solution (Gibco, Grand
Island, NY) which had been pre-equilibrated at 39~C in a
humidified atmosphere of 5~ CO2 in air.
Chimeras were generated by injection of both
epitheloid alkaline phosphatase negative and the three
dimensionally growing alkaline phosphatase positive ES
cells into the blastocoel cavity of New Zealand White
blastocyst (Table 2). A total of 287 New Zealand White
blastocysts were injected with 20 to 300 cells from the
GM3 cell line. Blastocyst injections with GM3 cells were
performed on a Zeiss inverted microscope with
differential interference optics at a magnification of
250X. Micromanipulations were performed with Narashige
micromanipulators as routinely employed for the mouse.
Between 5 and 10 embryos injected with GM3
cells were reimplanted into the proximal portion of each
uterine horn of a recipient New Zealand White doe using a
small incision and a sharpened glass pipette. This
recipient doe had previously received 75 IU of hCG, 14
hours after the time of hCG injection in the donors. The
reimplantation procedure was done under general
anesthesia with a ketamine/xylazine mixture.
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WO97/41209 PCT~P97/02323
Table 2: Production of chimeras after injection of ES
cells into the blastocoel cavity of New Zealand
White blastocysts
#blasts injected #ES cells passage #born(~) #chimeras(~)
76 20-30 22 30(41) 1(1)
124 50-100 15 31(25) 1(1)
87 100-300 18 6(7) 1(1)
Total 287 67(23) 3(1)
Percentages in parentheses are relative to numbers of
blastocysts injected.
This procedure resulted in an overall live
birth rate of 23~. Since the ES cell line used was
derived from the pigmented Dutch Belted strain, injection
of GM3 ES cells into blastocysts from a nonpigmented New
Zealand White strain will generate overt coat color
formation in chimeras. As judged by coat color, three
chimeric animals were obtained with one or more black
belts, typical for the Dutch Belted strain. The
percentage of chimerism varied between 10% to more than
50~, based upon contribution of the Dutch Belted
pigmented coat. One of these chimeras was male (Figure
2a), one female (Figure 2b) and one probably a
hermaphrodite. These results constitute the first proof
of principle that chimeric rabbits can be generated by
blastocyst injection of ES cell lines maintained in
culture and extensively (15 to 22 times) passaged. In
retrospect chimerism appeared to be attributable to the
three dimensionally growing alkaline phosphatase positive
ES cells, as further detailed below.
Indeed, the frequency of chimera formation
following injection of ES cells into the blastocoel
cavity was low - only 4~ of live born animals. This might
have been due to several factors, including 1) infrequent
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WO97/41209 PCT~P97/02323
~ incorporation of ES cells into the developing embryo due
to the prominent void space in the mature blastocyst into
which the ES cells were introduced, 2) an inherent incom-
patibility of Dutch Belted ES cell lines with New Zealand
White blastocysts, or 3) loss of pluripotency of the cell
lines. Attempt to increase the frequency of delivery of
ES cells to the developing inner cell mass by introducing
the ES cells directly onto or into the developing inner
cell mass, a prerequisite for incorporation into the
subsequent embryo, did not result in a higher frequency
or chimera formation. The use of Dutch White blastocysts
(originating from the Dutch Belted strain by a natural
point mutation) as recipients for ES cell injections also
did not generate chimeras, thus eliminating the
involvement of a strain barrier.
Additional experiments revealed that the
absence of chimera formation with ES cells maintained in
culture as described by Graves and Moreadith (1993) and
the low efficiency with ES cells cultured as described in
Example 1, starting from early passage GM3 cells was most
likely due to the absence in the former and the low
percentage in the latter of residual pluripotent ES
cells. This was revealed by staining for alkaline
phosphatase, which is present in undifferentiated cells,
but rapidly lost upon differentiation (Benham et al.,
1981). The occurrence of alkaline phosphatase positive
cells in the original cell line was less than 1~ after
passage 10 (Figure 1), al- though this frequency
cou~d be maintained under the improved culture conditions
described herein. It appears that the flat epithelioid
cell type (Figure 1) originally thought to represent
putative ES cells (Graves and Moreadith, 1993) are mostly
alkaline phosphatase negative, unable to incorporate in
the inner cell mass of the recipient blastocyst, and
unable to generate chimeric off-spring. Therefore new ES
cell lines were derived as described in Example 3.
EXAMPLE 3
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WO97/41209 PCT~P97/02323
12
Improved derivation of ES cells
Improved methods for derivation of ES cells
were developed which, in combination with the improved
cell culture conditions, allowed the generation of five
rabbit ES cell lines which consist of more than 80%
undifferen-tiated alkaline phosphatase positive cells
after 8 to 10 passages, as described below.
Superovulated Dutch Belted does were mated with
Dutch Belted bucks. The blastocysts were flushed from the
uterine horns on day 5.5 (postcoitus instead of 4 or 5
days postcoitus) and rinsed with Dulbecco's phosphate
buffered saline supplemented with 3% bovine serum albumin
(Cohn fraction V) plus 5% (v/v) antibiotic/antimycotic
solution. The blastocysts were kept in rabbit ES medium
(described above) at 39~C in a 5% CO2 incubator until
further manipulation. The mucin coat and zona pellucida
of the blastocysts were removed using acidified phosphate
buffered saline (pH=2.5) and 0.5% pronase in phosphate
buffered saline. The inner cell mass was prepared
manually out of the surrounding trophectoderm cells with
2 needles and placed individually in a 96 well culture
dish (plated with 12.5 days old, passage 1 mouse
embryonic fibroblasts with a density equivalent to 3 to 4
X 10 cells per 10 cm dish).
The explanted inner cell masses were refed
daily with the improved rabbit ~S cell medium described
above in which murine Leukemia Inhibitory Factor was
replaced by human or rabbit Leukemia Inhibitory Factor.
After 2 days, the inner cell mass outgrowth was easily
freed from remaining trophectoderm cells by gently
lifting the trophectoderm outgrowth with a beveled glass
pipet off the underlying feeder layer and by aspirating
it into the glass pipet.
Only ES-like colonies, characterised by three
dimensional growth were subsequently passaged onto new
culture dishes. Therefor the 96 well was selectively
trypsinized after 4 to 5 days with the trypsinization
medium described above, thus allowing the selective
CA 022s2s24 1998-10-22
W O 97141209 PCTIEP97/02323
13
passaging of ES cells and not of trophectoderm cells or
mouse embryonic fibroblasts. The three dimensional growth
typical of the improved rabbit ES cells has not been
noticed before and is apparently a novel characteristic
of such cell lines. Only with the improved culture
conditions and the use of 5.5 days old embryos, could
three dimensional undifferentiated ES cell colonies be
maintained at high frequency in culture. The ES cells
were passaged very gradually onto larger culture dishes
at 4 to 5 days intervals, to maintain the ES cells at a
very high density, another prerequisite to prevent
differentiation and loss of pluripotency. The feeder
densities on the subsequent culture dishes was maintained
at densities equivalent of 3 to 4 X 10 cells per 10 cm
dish.
The rabbit ES cell lines obtained by this
procedure (Figure 3) are more similar to pluripotent
murine ES cell lines than any of the rabbit ES variants
previously reported. The main characteristics are colony
growth in three dimensions, high refractility and a small
nuclear/cytoplasmic ratio. The most important
characteristics are that after 10 passages, 80 to 90% of
the ES cells remain undifferentiated as indicated by
their positive staining for alkaline phosphatase (Figure
3b), and negative staining for human cyto~eratin 18 and
mouse vimentin, which are known markers of
differentiation (Viebahn et al., 1988; Piedrahita et al.,
1990)~ These properties stand in major contrast with
those of the earlier putative ES cell line (Figure 1),
where less than 1% of the cells were undifferentiated as
judged by al~aline phosphatase positive staining. This
enormous increase in the percentage of undifferentiated
cells (from 1~ to 80-90~) in frequently passaged rabbit
ES cells, should markedly increase the efficiency of
chimeric rabbit generation by ES cell injection into
- blastocysts and allow the generation of chimeric rabbits
containing targeted genetic alterations.
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WO97/41209
14
REFERENCES
- Babinet C, Bordenave GR (1980)i Chimeric rabbits
from immunosurgically-prepared inner cell mass
transplantation. J Embryol Exp Morphoi 60:429-440.
- Benham FJ, Andrews FW, Knowles BB, Bronson DL,
Harris H (1981): Alkaline phosphatase isozymes as
possible markers of differentiation in human
testicular teratocarcinoma cell lines. Dev. Bio
88:279-287.
- Bongso A, Fong C-Y, Ng S-C, Ratman S (1994):
Isolation and culture of inner cell-mass from human
blastocysts. Hum Reprod 9:2110-2117.
- Bradley A (1987): Production and analysis of
chimeric mice. In: Teratocarcinomas and Embryonic
Stem Cells: A Practical Approach (Ed. Robertson EJ),
IRI Press Ltd., Oxford, 1987, pp. 113-15i.
- Capecchi MR (1989): Altering the genome by
homologous recombination. Science 244:1288-1292.
- Doetschman T, Williams P, Maeda N (1988):
Establishment of hamster blastocyst-derived
embryonic stem (ES) cells. Dev Biol 127:224-227.
- Du F, Giles JR, Foote RH, Graves KG, Yang X,
Moreadith RW (1995): Nuclear transfer of putative
rabbit embryonic stem cells leads to normal
blastocyst development. J Reprod Fert 104:219-223.
25 - Evans MJ, Notarianni E, Laurie S, Moor RM (1990):
Derivation and preliminary characterization of
pluripotent cell lines from porcine and bovine
blastocysts. Theriogenology 33:125-123.
- Gardner RL, Munro AJ (1974): Successful construction
of chimeric rabbit. Nature 250:146.
- Graves KH, Moreadith RW (1993): Derivation and
characterization of putative pluripotential
embryonic stem cells from preimplantation rabbit
embryos. Mol Reprod Dev 36:424-433.
35 - Iannaccone PM, Taborn GU, Garton RL, Caplice MD,
~renin DR (1994): Pluripotent embryonic stem cells
CA 022~2~24 1998-10-22
PCT~P97/02323
WO97/41209
- 15
from the rat capable of producing chimeras. Dev Biol
163:288-292.
- Johnson LV, Calarco PG, Siebert LS (1977): Alkaline
phosphatase activity in the preimplantation mouse
embryo. J Embryol exp Morph 40:83-89.
- Moustafa L (1974): Chimeric rabbits from embryonic
cell transplantation. Proc Soc Exp Biol 147:485-488.
- Nagy, A, J Rossant, R Nagy, W Abromow-Newerly, and
Roder JC (1993): Derivation of completely cell
culture derived mice from early-passage embryonic
stem cells Proc Natl Acad Sci USA 90:8424-8428.
- Nieman H, Strelchenko N (1994): Isolation of rabbit
embryonic stem (ES) cell like cells. Theriogenology
41:265.
15 - Notarianni E, Galli C, Laurie S, Moor RM, Evans MJ
(1991): Derivation of pluripotent, embryonic cell
lines from the pig and sheep. J Reprod Fert Suppl
43:25S-260.
- Notarianni E, Laurie S, Moor RM, Evans MG (1990):
Maintenance and differentiation in culture of
pluripotential embryonic cell lines from pig
blastocysts. J Reprod Fert 40:51-56.
- Piedrahita JA, Anderson GB, BonDurant RH (1990): On
the isolation of embryonic stem cells: Comparative
behaviour of murine, porcine and ovine embryos.
Theriogenology 34:879-891.
- Robertson EJ (1987): Embryo-derived stem cell lines;
In/ Teratocarcinomas and Embryonic Stem Cells: A
Practical Approach (Ed. Robertson EJ), IRI Press
Ltd., Oxford, 1987, pp. 71-112.
- Rubin EM, Barsh GS (1996): Biological insights
through genomics: mouse to man. J Clin Invest
97:275-280.
- Saito S, Strelchenko N, Nieman H (1992): Bovine
embryonic stem cell-like cell lines cultured over
several passages. Roux Arch Dev Biol 201:134-141.
- Strojeck M, Reed MA, Hoover JL, Wagner TE (1990): A
method for cultivating morphologically
CA 022~2~24 l998-l0-22
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WO 97/41209
16
undifferentiated embryonic stem cells from porcine
blastocysts. Theriogenology 33: 901-913.
- Sukoyan MA, Golubitsa AN, Zhelezova AI, Shilov AG,
Vatolin SY, Maximovsky LP, Andreeva LE, Mc Whir J,
Pack SD, Bayborodin SI, Kerkis AY, Kizilova HI,
Serov OL (1992): Isolation and cultivation of
blastocyst-derived stem cell lines from American
mink. Mol Reprod Dev 33: 418 - 431.
- Talbot NC, Caird ER jr, Vernon GP, Powell AM, Nel ND
(1993): Culturing the pig epiblast cells of the pig
blastocysts. In Vitro Cell Dev Biol 29A: 546-554.
- Thomson JA, Kalishman J, Golos TG, During M, Harris
CP, Becker RA, Hearn JP (1995): Isolation of a
primate embryonic stem cell line. Proc Natl Acad Sci
USA 92: 7844-7848.
- Viebahn C, Lane BE, and Ramackers FCS (1988) Keratin
and Vimentin expression in early embryogenesis of
the rabbit embryo. Cell Tissue Research 253: 553 - 562.
- Wilson JM (1996): Animal models of human disease for
gene therapy. J Clin Invest 97: 1138-1141.
- Yang X, Foote RH (1988): Production of chimeric
rabbi~s from morulae by a simple procedure. Gamete
Res 21: 345-351.
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17
LEGENDS TO THE FIGURES
~ Fig. 1:
Rabbit ES cells (GM3 l-ine), derived by Graves and
Moreadith.
A) phase contrast microscopy of the putative ES
cells revealing a flat epitheloid phenotype
B) alkaline phosphatase staining of the putative
ES cells, showing a very low percentage (< 1
percent) of alkaline phosphatase positive
cells (red).
Fig. 2:
A) male chimera with one black belt.
B) female chimera, showing several black belts.
The belts are typical for the Dutch Belted
strain from which the GM3 ES cell line was
derived.
FIg 3:
A) phase contract microscopy of a newly derived
cell line using the improved cell culture and
E~ derivation conditions described above.
B) alkaline phosphatase staining of a rabbit ES
cell line derived using the improved cel
culture and ES derivation conditions described
above. The new ES cell line is characterized by
three dimensional growth, high refractibility
and 80 to 90~ alkaline phosphatase positive
staining.
