WO2003054169A1

WO2003054169A1 - Compositions for the in vitro derivation and culture of embryonic stem (es) cell lines with germline transmission capability

Info

Publication number: WO2003054169A1
Application number: PCT/BE2001/000221
Authority: WO
Inventors: Luc Schoonjans
Original assignee: Thromb-X Nv
Priority date: 2001-12-21
Filing date: 2001-12-21
Publication date: 2003-07-03
Also published as: AU2002218893A1

Abstract

The present invention describes novel compositions for deriving, maintaining and growing pluripotent and germ-line competent mammalian embryonic stem cells. The compositions of this invention refers to compositions comprising a 1) conditioned medium of a cell line expressing limited amounts of Leukemia Inhibitory Factor (LIF), 2) conditioned medium from a cell line transfected with mammalian LIF and 3) a medium supplemented with recombinant rabbit LIF. The media of the present invention are used for the generation of pluripotent and germ line competent embryonic stem cells of mammals of which these cells were not obtained up to now. The present invention is also directed to a novel rabbit LIF and to nucleotides encoding the Rabbit LIF and methods for the expression of recombinant rabbit LIF in the Pichia pastoris expression system.

Description

COMPOSITIONS FOR THE IN VITRO DERIVATION AND CULTURE OF EMBRYONIC STEM ₍ES₎ CELL LINES WITH GERMLINE TRANSMISSION CAPABILITY

FIELD OF THE INVENTION

The present invention relates to novel compositions and their use for the derivation, maintenance and growth of pluripotent and germline competent embryonic stem (ES) cells. The invention further relates to the ES cells produced using the composition and to use of these ES cell lines for germline transmission and for the generation of genetically modified non-human animals.

BACKGROUND OF THE INVENTION

ES cell lines are cell lines isolated from the inner cell mass (ICM) of blastocyst-stage embryos, which under specific conditions can be maintained in culture for many passages, i.e. replating of cells onto new cell culture dishes at regular time intervals, without loss of their pluripotency. They maintain a normal karyotype and when reintroduced into a host blastocyst they can colonize the germline. Such cell lines may provide an abundance of pluripotent cells that can be transformed in vitro with DNA (see below), and selected for recombination (homologous or non-homologous) of exogenous DNA into chromosomal DNA, allowing stable incorporation of the desired gene. To date, germline transmission, i.e. the transmission of the ES genome to the next generation, has however only been achieved with ES cells of certain mouse strains.

Murine embryonic stem cells were first isolated in 1981. Since then, several ES cell lines have been established and they are now widely and successfully used for the introduction of targeted mutations or other genetic alterations into the mouse genome. Most of the germline-competent mouse ES cell lines that are in current use have been obtained in the 129 strain, and the remainder in a few other inbred strains (C57BL/6 and crosses with C57BL/6). Furthermore, ES cell lines are at best obtained in 30% of explanted blastocysts, even in the 129 strain, and success rates of around 10% appear to be closer to the norm.

The most commonly used approach to generate chimeric animals is to inject about 10-15 isolated ES cells into the blastocoel of a host blastocyst and to allow the cells to mix with the cells of the inner cell mass. The resultant chimeric blastocysts are then transferred to recipients for rearing. Alternatively diploid aggregation using very early (8-16 cell) stage embryos and tetraploid aggregation, can be used as hosts for ES cells. Briefly, ES cells are 'sandwiched' between early stage embryos devoid of their zona pellucida, cultured overnight and implanted into a foster mother. This technique can be performed under conditions yielding either chimeric or totally ES cell-derived offspring.

Although ES cell culture and chimera production have been technically improved over the years, the pluripotency of the ES cells is still often reduced after several passages, whereas completely ES cell-derived fetuses (by tetraploid aggregation) seem to have a markedly reduced survival after birth. Nagy et al., "Derivation of completely cell culture derived mice from early-passage embryonic stem cells" Proc Natl Acad Sci USA 1993; 90: 8424-8, used R1 ES cell lines derived from early passages with electrofusion derived tetraploid embryos to form aggregates and obtained mice which were entirely derived from ES cells. However, the Rl ES cells lost their totipotency upon extended culture in vitro, because no animal survived to adulthood from ES cells obtained from later than 14 passages. Moreover, even when early passage cells were used, many ES-tetraploid aggregates died before developing to term. Only 3.8% of transferred aggregates survived after caesarian section. The goal to obtain viable ES mice using later passage ES cells was not reached and the production of ES cell derived mice using genetically modified ES cells did not seem to be possible.

The inability of the present technology to yield viable offspring from ES cells of inbred mouse strains via tetraploid aggregation was recently confirmed in Eggan K, et al. "Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci 2001; 98: 6209-14. Genetic heterozygosity was found to be a crucial parameter influencing postnatal survival of offspring derived from ES cells by nuclear cloning or tetraploid embryo complementation. Pups derived from inbred ES cells by either method died perinatally with a phenotype of respiratory failure. In contrast, the great majority

(80-85%) of pups derived from F1 ES cells by either procedure survived to adulthood. In another study however, no clear correlation was found between the postnatal lethality of ES-cell-derived mice and the cell line used. Postnatal death occurred in all cell lines, including those with different genetic background. Thirty four completely ES-cell-derived newboms (3%) were obtained after transfer of

1037 tetraploid blastocysts injected with ES from all cell lines. Only thirteen mice (1 %) grew to adulthood (Amano T, Kato Y, Tsunoda Y. Comparison of heat-treated and tetraploid blastocysts for the production of completely ES-cell-derived mice. Zygote 2001 ; 9: 153-7).

Presumptive pluripotential ES cells have been isolated from a number of other species than mice, including hamster, pig, sheep, cattle, mink, rat, primate, human, chicken, marmoset, medakafish and man. In only a few instances (pig, chicken, medakafish), have these cell lines given rise to chimeras when reintroduced into blastocysts, but thus far none have given rise to germline transmission.

The isolation of pluripotential ES cell lines from preimplantation rabbit blastocysts was reported by Graves KH, Moreadith RW, "Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos", Mol Reprod Dev 1993; 36: 424-33. These ES lines were found to give rise to differentiated cell types, representative of all three germ layers (pluripotential by in vitro criteria). Recently these ES lines from the Dutch Belted strain were shown to be also capable of generating overt coat-color chimeras following injection into recipient New Zealand White blastocysts, demonstrating that the cells were pluripotential by in vivo criteria as well. However no germline transmission has been achieved. Additional experiments showed that the low frequency of chimera formation and absence of germline transmission probably was due to the loss of pluripotency of the ES cell line upon high passage number.

ES cells are maintained in an undifferentiated state by the presence of feeder layers producing various factor(s) that prevent the cells from differentiating. It has been shown that several cytokines are responsible for this effect: DIA/LIF (differentiation inhibitory activity/leukaemia inhibiting factor), interleukin-6 in combination with soluble interleukin-6 receptor, interleukin-11 , oncostatin M, ciliary neurotrophic factor and cardiotrophin. It is now possible to establish and maintain ES cells in culture in the absence of feeder cells but in the presence of such factors, at least for several passages. In species other than the mouse, ES cell technology is still under development and there are no published data reporting germ line transmission in any species other than mouse.

Recombinant Leukemia Inhibitory Factor (LIF) is presently routinely added to the culture medium used for the isolation of embryonic stem (ES) cells from mammalian embryos in vitro. This method is claimed in US 5,166,065, EP 0380646 and WO9001541 , based on a priority document AU1988 PI09644 dated August 4, 1988 (51-53). Recombinant murine or human LIF protein was purified and cDNA cloned on the basis of its ability to induce differentiation of the murine monocytic cell line M1 in mature macrophages with consequent reduced clonogenicity. The (recombinant) protein and cDNA's (the murine and human variants) are claimed in a.o. US 5,187,077 (and several continuations in part to up to US 6,261 ,548 issued 17 July 2001) and EP 285448, based on a priority document of AU1987 PI1209 dated April 2, 1987.

Subsequent work has established the identity of LIF with earlier purified proteins and/or biological activities. The work of Hozumi et al. during 1980-1986 led to the purification to homogeneity of Factor D which stimulated the differentiation and inhibited the proliferation of the murine monocytic cell line M1 , Tomida M, Yamamoto-Yamaguchi Y, Hozumi M. Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium or mouse fibroblast L929 cells. J Biol Chem 1984; 259: 10978-82). The Factor D cDNA was subsequently shown to be identical to that of LIF (Lowe DG, Nunes W, Bombara M, McCabe S, Ranges GE, Henzel W, Tomida M, Yamamoto-Yamaguchi

Y, Hozumi M, Goeddel DV. Genomic cloning and heterologous expression of human differentiation-stimulating factor. DNA 1989; 8: 351-9). The use of LIF in the culture medium of ES cells was preceeded by work on the inhibition of the differentiation of murine embryonic stem cells by DIA (differentiation inhibiting activity) secreted by Buffalo rat liver cells. Subsequently the identity of DIA and LIF was established at the cDNA and protein level (Smith AG, Heath JK, Donaldson

DD, Wong GC, Moreau J, Stahl M, Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988; 336: 688-90; Smith AG, Nichols J, Robertson M, Rathjen PD. Differentiation inhibiting activity (DIA/LIF) and mouse development. Devel Biol 1992; 151 : 339-51.).

Advances in recombinant DNA technology over the last decade have greatly facilitated the isolation and manipulation of genes, to the point where any conceivable novel construct can be engineered, such as by fusing the promoter of one gene to the coding sequence of another, or by site-directed mutagenesis. Likewise, advances in embryo manipulation have facilitated the transfer of these novel exogenous genes into endogenous chromosomal DNA, generating transgenic animals. Transgenic animals can be generated either by injection of DNA into the pronucleus of zygotes, by introduction of (genetically manipulated) pluripotent embryonic stem (ES) cells into host "embryos", and more recently by nuclear transfer with stably transfected somatic donor cells into enucleated oocytes.

The review of the current technology shows that there is a need for economic compositions that provide ES cells which remain pluripotent and germ line competent after prolonged passaging. There is also a need for the generation of transgenic mice of strains with different genetic background and for the generation of other non human transgenic mammals. These transgenic animals could be useful for the study of the biological effects of identified genes, for the pharmaceutical production of therapeutic gene products, for the generation of "improved" live stock, etc. SUMMARY OF THE INVENTION

The present invention is directed to novel and superior compositions for deriving, maintaining and growing pluripotent and germline competent mammalian embryonic stem cells and the use of these compositions. In a first aspect of the invention, the composition for deriving, maintaining and growing pluripotent and germline competent human or non-human mammalian embryonic stem cells is provided comprising a conditioned medium from certain cells and wherein the composition contains less than 2 ng/mi of leukemia inhibitory factor (LIF) as assayed by an immunoassay, e.g. an ELISA using antibodies which cross-react with rabbit and human LIF such as the mouse monoclonal antibody clone 9824.11 raised against human LIF. Preferably, the concentration of LIF is less than 1 ng/ml, more preferably less than 0,5 ng/ml, still more preferably less than 0,2 ng/ml and most preferably the concentration of LIF is less than 0,02 ng/ml LIF as determined with said assay. The composition can include a basic cell culture medium such as, but not limited to, a medium comprising high glucose DMEM with further optional addition of one or more of the compounds selected from non-essential amino acids, glutamine, a reducing agent and fetal, newborn or adult serum such as fetal bovine serum. The cells used for conditioning the medium are cells such as immortal fibroblast cell line such as the rabbit fibroblast cell line Rab9 (ATTC CRL-1414).

In another aspect of the invention, the composition for deriving, maintaining and growing pluripotent and germline competent human or non-human mammalian embryonic stem cells is a composition comprising a conditioned medium from certain cells transfected with a nucleotide sequence encoding LIF. Said nucleotide sequence encodes for mammalian LIF and preferably encodes for rabbit LIF. Said nucleotide sequence can be a cDNA sequence but is preferably a genomic sequence of the mammalian LIF. The transfected cell line is preferably stably transfected with the LIF encoding nucleotide sequence. The cells used for transfecting a nucleotide sequence encoding for LIF can be any mammalian cell but is preferably a fibroblast cell, more preferably a rabbit fibroblast cell line and most preferably the rabbit Rab9 (ATCC CRL 1414). The transfected cells used in this invention are rabbit Rab9 fibroblast cells which are stably transfected with a genomic sequence encoding for rabbit LIF. Such a cell line has been deposited with the Belgian Coordinated Collection of Microorganisms, Belgium under accession number LMBP 5479CB. The composition can include a basic cell culture medium such as, but not limited to, a medium comprising high glucose

DMEM with further optional addition of one or more of the compounds selected from non-essential amino acids, glutamine, a reducing agent and newborn or adult serum and a fetal serum other than bovine fetal serum such fetal horse serum , fetal goat serum, fetal sheep serum.

In a third aspect of the invention, the composition for deriving, maintaining and growing pluripotent and germ-line competent human or non-human mammalian embryonic stem cells is a composition comprising a conditioned medium from certain cells and wherein the composition is supplemented with rabbit LIF, a protein with at least 95% similarity to rabbit LIF or a functional derivative thereof. Said protein may be expressed in a yeast suitable for protein expression such as the methylotrophic yeast Pichia pastoris and wherein the nucleotide sequence encoding for said protein is optionally adapted in order to obtain an optimized sequence for use in the yeast expression system. The rabbit LIF disclosed in this invention is produced by the Pichia pastoris strain which has been deposited at the Belgian Coordinated Collection of Microorganism, Belgium under accession number MUCL-49925.

The composition can include a basic cell culture medium such as, but not limited to, a medium comprising high glucose DMEM with further optional addition of one of the compounds selected from non-essential amino acids, glutamine, a reducing agent and fetal, newborn or adult serum such as fetal bovine serum.

The present invention discloses the use of these compositions for the generation of multipotent or pluripotent embryonic stem cells of non human mammals such as but not limited to mouse and more specifically to Mus musculus strains selected from the group 129/SvEv, C57BL/6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola, C3H/HeN, C57BL/6JOIa,

FVB/ and Swiss Webster genetic background.

The present invention relates further to multipotent or pluripotent embryonic stem cells of human or non human mammals such as but not limited to mouse and more specifically to mus musculus strains selected from the group of 129/SvEv,

C57BL/6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N,

DBA/1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster genetic background which are obtained by culturing them for at least one passage in the compositions of the present invention. The present invention relates further to transgenic non human mammals such as but not limited to mouse and more specifically to mus musculus strains selected from the group of 129/SvEv, C57BL/6N, C57BL/6J-HPRT, BALB/cAnN,

CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and

Swiss Webster genetic background which are obtained by culturing the embryonic stem cells used for the generation of said animals for at least one passage in the compositions of the present invention.

The present invention relates further to methods for producing the compositions, embryonic stem cells and transgenic animals disclosed in this invention. The present invention discloses the use of media conditioned by rabbit cell lines for the generation of embryonic cells and transgenic animals and is applicable to other non human mammals such as non-human primates, pigs, cows, horses, goats, sheep, cats, dogs rabbits and rodents other than the mouse Mus musculus.

The present invention further discloses methods to obtain embryonic stem cell lines for 30 or more percent of explanted blastocysts.

The present invention also discloses methods to obtain adult progeny via aggregation cells with embryonic stem cells that have been cultivated for at least

16 passages.

The present invention is particularly directed to novel compositions for deriving, maintaining and growing pluripotent and germ-line competent mammalian embryonic stem cells. The compositions include a basic cell culture medium such as, but not limited to, a medium comprising high glucose DMEM, non-essential amino acids, glutamine, a reducing agent and fetal bovine serum, or equivalents thereof, which are preconditioned by a stable cell line such as, but not limited to, the rabbit cell line Rab9 (ATCC CRL-1414) or equivalents thereof, which secrete essential elements for growth and self renewal of ES cells. It has been found that this conditioned media with low amounts and even in the absence of LIF supports self-renewal of ES cells and allows ES ceil derivation. Maintenance of the undifferentiated state, as evidenced by morphological and surface marker criteria (presence of alkaline phosphatase and absence of vimentin and cytokeratin), was superior to that observed with standard cell culture media to which murine LIF or rabbit LIF was added.

To this composition purified recombinant Leukemia Inhibitory Factor (LIF) can optionally be added, preferably rabbit LIF (Rab-LIF) disclosed in the present invention, or alternatively commercially available LIF. Antibiotics, such as penicillin/streptomycin, and insulin, may also be included in the composition. The present invention is also directed to a novel rabbit LIF (Rab-LIF) which maintains ES cells undifferentiated in in vitro culture and to nucleotides encoding the Rab- LIF. Other ingredients may optionally be included in the ES medium, such as interieukins, oncostatins, neurotrophic factors, stem cell factors and fibroblast growth factors. Specific examples of these factors are human Interleukin 11 , human oncostatin M, human ciliary neurotrophic factor, cardiotrophin, Interleukin 6 with its specific receptor and human stem cell factor. The invention further relates to the use of these ES cell lines for germline transmission and for the generation of genetically modified non-human animals.

The present invention will now be described with reference to the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the nucleotide sequence [SEQ. ID.: NO 1] of rabbit LIF (Rab-LIF) cDNA along with the peptide sequence thereof [SEQ. ID.: NO 2.

Figure 2 shows the nucleotide [SEQ. ID.: NO 3] and amino acid [SEQ. ID.: NO 4] sequence of rabbit LIF cDNA optimized for expression in Pichia pastoris.

Figure 3: Initial outgrowth (passage 0) of attached blastocysts from C57BL6/N mouse, when cultured in A) enriched basic medium with added murine

LIF (1 ,000 lU/ml); B) enriched basic medium with added Rab-LIF (10-20 ng/ml); C) basic medium conditioned on Rab9 fibroblast cells; D) basic medium conditioned on the Rab9 #19 fibroblast cell line; E) enriched basic medium.

Figure 4: Morphology of ES cell colonies after 1 passage in A) enriched basic medium with added murine LIF (1 ,000 lU/ml); B) enriched basic medium with added Rab-LIF C) basic medium conditioned on Rab9 fibroblast cells; D) basic medium conditioned on the Rab9 #19 fibroblast cell line; E) enriched basic medium.

DEFINITIONS

Leukemia Inhibitory Factor (LIF) as used herein refers to a mammalian protein, originally cloned and sequenced by Gearing et al. (1987) EMBO J. 6, 3995-4002 which enables the derivation, growth and maintenance of undifferentiated embryonic stem cells derived from the inner cell mass of blastocysts. It refers also to splice variants of LIF and to variants of LIF wherein one or more amino acids are mutated, inserted or deleted with the restriction that the variant LIF is at least 95% identical to wild type LIF and that it enables the derivation, growth and maintenance of undifferentiated embryonic stem cells derived from the inner cell mass of blastocysts.

Functional protein fragment of LIF as used herein refers to a LIF protein with N or C terminal deletions which still enable the derivation, growth and maintenance of undifferentiated embryonic stem cells derived from the inner cell mass of blastocysts. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of the disclosed compositions for the derivation, maintenance and growth of pluripotent and germline-competent mammalian embryonic stem (ES) cell lines, as exemplified in mouse strains. These improved culture conditions can be used to generate stable murine ES cells from many different genetic backgrounds, with superior potential for germline transmission. This technology is also applicable to other no human mammalian species (rabbits, pigs, cattle, etc.) and can form the basis for targeted transgenesis with gain-of-function or loss-of-function in non murine species. The compositions consist of a basic cell culture medium, which is preconditioned by a stable cell line, that secretes essential elements for growth and self renewal of ES cells. To these compositions purified recombinant Leukemia Inhibitory Factor (LIF) can optionally be added, e.g. in low amounts.

In one embodiment, the basic cell culture medium is high glucose Dulbecco's modified Eagle's medium DMEM, with non-essential amino acids, glutamine, beta-mercaptoethanol and fetal bovine serum, and the stable cell line is a rabbit fibroblast-like cell line Rab9 (ATCC CRL-1414). The optional addition of LIF preferably consists of the newly disclosed rabbit LIF (Rab-LIF) which helps maintain ES cells undifferentiated in in vitro culture, and which is obtained by expression of an optimized cDNA sequence in Pichia pastoris.

LIF secreted by cells into the medium can be determined via a quantitative immunossay such as linked immuno sorbent assay (ELISA), a radioimmunoassay (RIA), an immuno radio metric assay (IRMA), a fluorescent immunoassay (FIA), a chemiluminescent immuno assay (CLIA) or an electro chemiluminescent immuno assay (ECL) using antibodies against LIF. In this invention an ELISA using a monoclonal antibody with reactivity against rabbit LIF, such as the mouse monoclonal antibody Clone 9824.11 raised against human LIF, is preferred as a reference. An example of such an assay is the Quantikine Human LIF immunoassay (Cat No DLF00, R&D Systems Minneapolis, MN, USA). The compositions of the invention may have a varying amount of additional constituents provided that their amount is sufficient to maintain ES cells undifferentiated for prolonged periods in culture

The composition of the invention comprises conditioned medium of the fibroblast-like cell line in a medium selected but not limited to Phosphate Buffered

Saline (PBS), Dulbecco's Modified Eagle Medium (DMEM), Iscove's Modified,

Dulbecco's medium, McCoy 5A medium, Minimal Essential Media Eagle (MEM),

RPMI 1640, Medium 199, MCDB Medium, RPMI, Glasgow minimum Essential

Media Eagle (GMEM), DMEM/F-12 Media, Hams F-10 Nutrient mixture, Leibovitz's L15 Media, CMRL Media, BGJb Medium, Basal Medium Eagle (BME), Brimster's BMOC-3 Medium, William's Media E and McCoy's Media or adaptations thereof. To the composition further a reducing agent such as beta-mercapto ethanol or dithiothreitoi.

In the composition containing conditioned medium of cells which are not transfected with DNA encoding LIF, the medium is supplemented with adult, newborn or fetal serum. In the composition with conditioned medium from cells transfected with DNA encoding LIF, the serum can be adult or newborn mammalian serum or non bovine fetal serum such as horse, goat and sheep fetal serum. A preferred example of a composition of the invention comprises per liter conditioned medium of the fibroblast-like cell line, an added volume of 50 to 120, preferably 80 ml of fetal bovine serum, 10 to 25, preferably 17 ml non-essential amino acids, 2 to 8, preferably 5μi β-mercaptoethanol, 0.5 to 2.5, preferably 1.25 ml insulin, and 80 to 130 ml basal ES cell medium. Preferably, the basal ES cell medium consists of 400 to 600, preferably 500 ml DMEM high glucose, 0 to 15, preferably 13 ml penicillin/streptomycin, 10 to 15, preferably 13 ml non essential amino acids, 10 to 15, preferably 13 ml glutamine, 5 to 10, preferably 6.3 μl β-mercaptoethanol, 50 to 100, preferably 70 ml fetal bovine serum, neutral pH of preferably 7.4. The invention further relates to processes to derive and to culture mammalian ES stem cells to obtain pluripotent and germline-competent ES cells, wherein the culturing of the mammalian ES stem cells is at least partially performed in a composition according to the invention and described above. Such a process comprises the steps of: a) culturing cells of blastocyst stage embryos; b) culturing isolated inner mass cells; and c) passaging the inner mass cells periodically in a composition of the invention. Preferably, the inner mass cells are periodically passaged for at least 8 times. The process may further comprise the step of producing transgenic animals.

According to a further aspect thereof the invention relates to embryonic stem (ES) cell lines with germline transmission capability. The cell line is preferably a murine cell line, but other animal cell lines are also possible. In case of a murine cell line, the cell line can be derived from cells or tissues of 129/SvEV, C57BL/6N, C57BIJ6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 CaOla, C3H/HeN, C57BI/6Ola, FVB/N or Swiss Webster genetic backgrounds. The murine cell lines preferably have a germline transmission capability after 11 or more passages.The embryonic stem (ES) cell lines of the invention are characterized by three dimensional colony formation, positive staining for alkaline phosphatase and negative staining for cytokeratin 18 and vimentin after more than 10 passages. These embryonic stem (ES) cell lines may be used in the generation of chimeric or ES cell derived animals, in the gene alteration by homologous or non-homologous recombination, in the generation of animals with gene alteration via germline transmission, for the generation of chimeric animals, for the generation of chimeric animals following blastocyst injection into recipient blastocysts or diploid or tetraploid embryo aggregation, or nuclear transfer, for the study or isolation of (novel) genes or for the expression or overexpression of genes.

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 variations and improvements will be obvious to those skilled in the art. EXAMPLES EXAMPLE 1 : Production of recombinant Rabbit Leukemia Inhibitory Factor (Rab- JJF1

The above-referenced method and ES cell medium can optionally include

LIF, preferably recombinant Rab-LIF, to help maintain ES cells undifferentiated and capacity to produce germline transmission. In Figure 1 , the nucleotide sequence of Rab-LIF is provided along with the peptide sequence thereof.

Rab-LIF may be prepared by a number of methods, typically by one of the many molecular biological tools, i.e., expression systems, available to biologists. In such a case, DNA molecules encoding Rab-LIF protein may be operably linked to

DNA molecules encoding a transcription promoter and terminator to create an expression cassette. The DNA molecule containing the promoter, terminator and Rab-LIF-encoding DNA may then be introduced into a cell for production of the Rab-LIF protein. Preferably, a DNA encoding a secretion signal is operably linked to the Rab-LIF-encoding DNA in such a manner that active Rab-LIF is secreted from the cell. The promoter may be constitutive, inducible or tissue-specific, the choice of which typically depends upon the cells in which it is desired to produce the Rab-LIF protein, and under which conditions. As described herein in Example 1 , recombinant Rab-LIF is produced in Pichia pastoris and is operably linked with the yeast alpha factor secretion signal, and the alcohol oxidase 1 (AOX1) promoter. Alternatively, the Rab-LIF-encoding DNA may be operably linked to appropriate promoters, enhancers or terminators (collectively, "control sequences") for expression in prokaryotic cells or higher eukaryotic cells such as mammalian cells and insect cells.

Recombinant Rab-LIF was produced using the methylotrophic yeast Pichia pastoris expression system from Invitrogen (Carlsbad, CA). The Rab-LIF gene was isolated from a rabbit genomic library Lambda DASH II (Stratagene, # 945950) and the cDNA encoding the mature Rab-LIF protein was assembled by spliced overlap extension polymerase chain reaction (SOE-PCR) using standard procedures.

The Rab-LIF cDNA was used as template in consecutive PCR and SOE- PCR reactions to optimize the gene for expression in P. pastoris, by modifying the codon usage. The nucleotide sequence of this Rab-LIF optimized cDNA is shown in figure 2.

The primer in the PCR reaction was designed to allow precise in frame fusion of the mature Rab-LIF sequence with the alpha factor secretion signal in the pPICZ vector (Invitrogen). This allows the isolation of the recombinant protein from transformed Pichia pastoris culture supernatant. This primer introduced also an extra alanine codon (underlined in the sequence above) in front of the mature

Rab-LIF coding sequence to facilitate the Kex2 processing of the alpha factor secretion signal. The LY-RLIF-PD primer contained a Notl site. The product was purified, digested with Xhol and Notl, and cloned in the corresponding sites of the vector pPICZα, yielding pPICZα-RLIF100. In this vector, expression of the Rab-LIF is directed by the strong alcohol oxidase I (AOX1) promoter.

Prior to transformation in P. pastoris, the vector pPICZα-RLIF100 was linearized by digestion with BstX1, cutting in the 5' AOX1 untranslated sequence, to allow stable integration of the expression module in the AOX1 chromosomal locus. All the yeast manipulation was further performed as recommended by the supplier. The P. pastoris transformant X33 (RbL) was finally selected as Rab-LIF yeast expression strain and deposited in the Belgian Coordinated Collections of Microorganisms (#42925 BCCM Collection, MUCL, place Croix du Sud 3, B-1348 Louvain-La-Neuve, Belgium).

It should be noted that the expression cassette described above is stably integrated in the chromosome. The X33 (RbL) strain was deposited in the BCCM Collection on July 5, 2000. The optimized nucleotide sequence for expression in Pichia forms a part of the present invention. The nucleotide sequence and amino acid sequence of the Rab-LIF cDNA which has not previously been reported are shown in Figure 1. The nucleotide sequence was determined according to the standard method of Sanger et al.

The Rab-LIF nucleic acid sequence of the mature protein was manually compared to the human LIF sequence (Gough et al. 1988, accession M63420 & J05436 and mouse LIF sequence (Gearing et al. 1987, accession M63419 & J05435.) A homology of 90% was found between the nucleic acid sequence of human and rabbit LIF. A homology of 77% was found between the nucleic acid sequence of mouse and rabbit LIF. The corresponding homologies of the optimized cDNA for expression in Pichia pastoris was 61% with murine and 70% with human LIF.

EXAMPLE 2: Production of ES cell culture medium preconditioned with Rab9

In one embodiment of the present invention, basic ES cell medium, conditioned by confluent monolayer cultures of the Rab9 fibroblast cells, is collected for 4 consecutive days and the conditioned media are pooled for use in ES cell culture. Each day 15 cm Petri dishes are refreshed with 25 ml of basic ES medium. After 4 days each 15 cm Petri dish is split at a ratio of 1 to 4. The first day after splitting, the medium is discarded. To 1 liter of conditioned basic ES medium (from the mixture of the 4 collection days), 80ml fetal bovine serum, 17ml non- essential amino acids, 20ml glutamine, 6.3μl β-mercaptoethanol, 1.25 ml insulin and 80ml basal medium is added and the pH is adjusted to 7.4. The basic medium was composed of: 500 ml DMEM high glucose, 70 ml fetal bovine serum, 13ml penicillin/streptomycin, 13ml glutamine, 6.3μl β-mercaptoethanol, and 13ml non- essential amino acids. Enriched basic medium is basic medium to which another 4% (v:v) fetal bovine serum is added.

In other embodiments, the production of conditioned basic ES medium can be scaled up using standard procedures such as roller bottles, cell factories or bioreactors.

EXAMPLE 3: Derivation and culture of murine embryonic stem (ES) cells

1. Mouse strains and ES cells

ES cells can be derived amongst others from the following commercially available mouse strains: 129/SvEvTaconic (Taconic, Germantown, NY, USA); C57BU6NTacfBr (Taconic); BALB/cAnNTacfBr (Taconic); DBA/2NTacfBR (Taconic); C3H/HeNTac MTVfBe (Taconic); FVB/NTacfBR (Taconic);

Tac:(SW)fBR, Swiss Webster (Taconic); 129/SvJ (The Jackson Laboratory, Bar

Harbor, Maine, USA); C57BLJ6J-HPRT <B-M3> (The Jackson Laboratory);

C57BL/6JOIaHsd (Harlan, Indianapolis, Indiana, USA); CBA/CaOlaHsd (Harlan); DBA/1 OlaHsd (Harlan).

2. Derivation of murine ES cells

ES cells can be derived from 3.5-4.5 days old blastocyst stage murine embryos, which can be collected and plated individually on a 96 well dish covered with a mitotically arrested mouse embryonic fibroblast feeder monolayer. The blastocysts are allowed to attach to the monolayer, and refed every day with conditioned ES cell medium of the present invention (see Example 2), with basic ES medium with or without addition of murine LIF or Rab-LIF, or with ES cell medium conditioned with the Rab9#19 cell line which secreted endogenous Rab- LIF (see below).

After 5-6 days in culture, the inner cell mass (1CM) outgrowth is selectively removed from the (remaining) trophectoderm and replated after trypsinization with trypsin-EDTA on a 96 well dish with mitomycin arrested murine fibroblasts. Subsequently the ES cells are gradually plated on larger culture dishes. ES cells can remain undifferentiated for more than 20 passages by using conditioned ES cell medium of the present invention.

Fibroblast feeder layers can be obtained from murine embryos of 12.5 days post-coitus pregnant mice. The mice are sacrificed, and the uteri collected and placed in a petri dish containing phosphate buffered saline (PBS). The embryos are dissected out of the uterus and all membranes removed. The embryos are transferred into a new dish containing PBS, the head and all internal organs removed and the carcasses washed in PBS to remove blood. The carcasses are then minced using 2 insulin syringes into cubes of 2 to 3 mm in diameter, and incubated in Trypsin-EDTA/MEM solution (10/90 V/V) at 4°C for 2 hrs. The suspension is then incubated at 37°C for 15 min, a single cell suspension made using a 5 ml pipette, and plated at 5 x 10⁶ cells per 180 mm petri dish in 25 ml

Feeder Medium.

Feeder Medium consisted of 500 ml Dulbecco's Minimal Essential Medium

(DMEM), 10% fetal calf serum (FCS), 13 ml penicillin/streptomycin, 13 ml glutamine, 13 ml non-essential amino acids, 2.3 μi β-mercaptoethanol. The medium is changed after 24 hr to remove debris. After 2 to 3 days of culture the fibroblasts reaches a confluent monolayer. The plates are then trypsinized, replated on 2 petri dishes, and, when confluent, the cells of each plate are frozen in

2 vials, kept at -80°C overnight and transferred to liquid nitrogen the next day.

3. Culture of ES cells

ES cells are grown to subconfluency on mouse embryonic fibroblasts mitotically arrested with mitomycin. Culture dishes are kept at 39°C in a humidified atmosphere of 5% C0₂ in air. The ES cells are passaged every 2-3 days onto freshly prepared feeder dishes. The ES cells are fed every day with the conditioned

ES cell medium.

4. Comparison

In one aspect of the invention, the blastocysts were obtained from the natural matings of C57BL/6N TacfBr (Taconic) mice. The blastocysts were cultured with: a) enriched basic medium (see below); b) enriched basic medium with added murine LIF (1000IU/ml); c) enriched basic medium with added Rab-LIF (10ng/ml); d) enriched basic medium with added Rab-LIF (20ng/ml); e) basic medium conditioned on Rab9 fibroblast cells according to example 2; or f) basic medium conditioned on the Rab9 #19 fibroblast cell line (see below).

The basic medium was composed of: 500 ml DMEM high glucose, 70 ml fetal bovine serum, 13ml penicillin/streptomycin, 13ml glutamine, 6.3μl β- mercaptoethanol, and 13ml non-essential amino acids. Enriched basic medium is basic medium to wich another 4% (v:v) fetal bovine serum is added. The basic medium conditioned by the Rab9 fibroblast cells is obtained as illustrated in Example 2. To 1 liter of conditioned basic ES medium (from the mixture of the 4 collection days), 80ml fetal bovine serum, 17ml non-essential amino acids, 20ml glutamine, 6.3μl β-mercaptoethanol, 1.25 ml insulin and 80ml basal medium is added and the pH is adjusted to 7.4. This conditioned medium contains unmeasurable level (less than 20 pg/ml) of Rab-LIF as determined with the ELISA for human LIF of R&D Systems (Minneapolis, MN, USA).

Basic medium, conditioned by the Rab9#19 fibroblast cells, is collected for 4 consecutive days as described for Rab9 in Example 2. To 1 liter of conditioned basic ES medium (from the mixture of the 4 collection days), 80ml fetal bovine serum, 17ml non-essential amino acids, 20ml glutamine, 6.3μl β-mercaptoethanol, 1.25 ml insulin and 80ml basal medium is added and the pH is adjusted to 7.4. Rab9#19 are Rab9 fibroblast cells which have been stably transfected with the rabbit Leukemia Inhibitory Factor gene and which secrete up to 30 ng/ml/day of Rab-LIF in the medium as determined with the ELISA for human LIF of R&D Systems (Minneapolis, MN, USA).

The blastocysts are allowed to attach to the feeder layer. The culture medium is refreshed every day. After approximately 1 week in culture, the ICM outgrowth is removed from the trophectoderm and after trypsinization passed onto a new 96 well dish covered with a feeder layer of mitomycin C arrested murine fibroblasts. The ES cells are subsequently gradually passed onto larger culture dishes with a feeder layer. After 5 to 10 passages, the number of established ES cell lines is counted for each of the culture conditions. The undifferentiated character of the established ES cell lines is determined by immunochemical staining for the presence of alkaline phosphatase (Vector Laboratories Inc., Buriingame, CA), or for the absence of vimentin and cytokeratin (both Dako A/S, Denmark). Only ES cell lines which consist for more then 90% of undifferentiated cells are maintained in culture.

Enriched basic medium alone did not allow ES cell derivation. ICM outgrowth rapidly differentiated before or during the first passage (Figure 3E and Figure 4E). Table I: Efficiency of murine C57BL6/N ES cell derivation

When either murine LIF or RabLIF was added to the enriched basic medium, the efficiency of ES cell derivation increased to approximately 25% after 3 passages. The efficiency of ES cell derivation with either murine or rabbit LIF was comparable. The efficiency of ES cell derivation increased to approximately 50% when Rab9 conditioned medium was used in the absence of endogenous or added LIF. A similar ES cell derivation efficiency was obtained when basic medium was conditioned with the Rab9#19 cell line which secreted endogenous Rab-LIF.

When blastocysts were cultured in basic medium conditioned on Rab9 or Rab9#19 fibroblast cells (Figure 3C and D), there was a facilitated outgrowth of inner cell mass cells. In enriched basic medium with added murine LIF or Rab-LIF, partial differentiation of the inner cell mass or outgrowth of throphectodermal cells was seen (cfr Figure 3A and B).

Already after one passage, a difference was observed in the morphology of

ES cell colonies in different culture media. Enriched basic medium with added murine or Rab-LIF (Figure 4A and B) gave rise to rather flat ES colonies, while the use of basic medium conditioned on Rab9 or Rab9#19 fibroblast cells (Figure 4C and D) resulted in three-dimensional ES cell colonies. When basic medium was used all cells were differentiated after 1 passage (Figure 4A).

EXAMPLE 4: Generation of chimeric and ES cell derived animals The ability of the ES cells to colonize the germline of a host embryo can be tested by injection of these ES cells into host blastocysts, or by their aggregation with moruia-stage diploid embryos or 4-celled tetraploid embryos, and implanting these chimeric preimplantation embryos into pseudopregnant foster recipients according to standard procedures. The resulting chimeric offspring can be test bred for germline transmission of the ES cell genome.

1. Blastocyst injection ofES cell clones

ES cells of mouse strains with a coat colour (C57BLJ6J-HPRT, DBA/2N, DBA/1 Ola) can be injected into host blastocysts of albino Swiss Webster mice. ES cells of mouse strains with a white or cream coat color (Swiss Webster, 129/SvJ, BALB/cAnN, and FVB) can be injected into host blastocysts of black C57BL/6N mice. This allows easy identification of ES cell contribution. All ES lines tested resulted in chimeric offspring with germline capability.

2. Diploid aggregation of ES cell clones

The diploid aggregation method can be executed as follows. Swiss Webster (albino coat colour) females are superovuiated with pregnant mare serum gonadotropin followed 44-48 hrs later by 5 units human chorionic gonadotropin. The oviducts of superovuiated and mated Swiss Webster mice are flushed 2.5 days after copulation to collect late 8-cell stage diploid embryos. All ES cell lines tested are derived from mice strains with a coat colour, facilitating identification of chimeric offspring.

Zonae pellucidae of these 8-cell stage diploid embryos are removed by treatment with acid Tyrode's buffer. The zona-free embryos are washed and placed in M16 medium. Aggregation is performed between one 8-cell stage diploid embryo and a clump of ES cells. The aggregates are cultured in micro drops of

M16 until the blastocyst stage before they are reimplanted into the uterus horns of

2.5-day pseudopregnant Swiss Webster females.

Chimeric pups are identified by the presence of a dark (= non albino) colour, which originated from an ES cell contribution. The percentage of chimerism

(portion of the newborn pup, originating from the ES cells) is visually identified by judging the percentage of dark coat (originating from the ES cells) compared to the white coat (originating from the albino Swiss Webster embryo).

3. Tetraploid aggregation ofES cell clones

Completely ES cell derived embryos can be generated via aggregation of the ES cells with tetraploid host embryos. 2-celled embryos are electrically fused, and subsequently aggregated as 4-celled tetraploid embryos with the ES cells to form chimeric embryos, which are then implanted in pseudopregnant recipients. The ES cells (almost) exclusively contributes to the development of the embryo proper, and the tetraploid cells to that of the extra embryonic membrane.

In order to distinguish between the ES and tetraploid cells, host embryos (used for aggregation) are derived from the ROSA26 strain, which expresses LacZ ubiquitously and throughout the entire development and adulthood. The oviducts of superovuiated and mated ROSA26 mice are flushed 36 hrs after treatment with human chorionic gonadotropin to collect late two-cell stage embryos.

Claims

1. A composition for the derivation, maintenance or growth of pluripotent embryonic stem cells of mammals for at least 10 passages as undifferentiated cells comprising a conditioned medium of cells, said composition characterised in that it contains less than 2 ng /ml Leukemia Inhibitory Factor (LIF).

2. A composition according to claim 1 , characterised in that it contains less than 0,2 ng / ml Leukemia Inhibitory Factor (LIF).

3. A composition according to claim 1 , characterised in that it contains less than 0;02 ng / ml Leukemia Inhibitory Factor (LIF).

4. A composition according to any of claims 1-3 wherein the composition is suitable for stem cells of a non-human mammal selected from the group of primates, pigs, cows, horses, goats, sheep, cats, dogs or rabbits.

5. A composition according to any of claims 1-3 wherein the composition is suitable for stem cells of a non-human mammal selected from the group of rodents.

6. A composition according to claim 5 wherein the non-human mammal is Rattus sp.

7. A composition according to claim 5 wherein the non-human mammal is Mus sp.

8. A composition according to claim 7 wherein the non-human mammal is a Mus musculus mouse strain of any of the strains with a genetic background background selected from the group of 129/SvEv, C57BL/6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster.

9. A composition according to claim 7 wherein the non-human mammal is selected from the group of Mus musculus strains with a genetic background of

C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster

10. A composition according to claim 7 wherein the non-human mammal is a mouse with a genetic background of 129/SvEv or with a genetic background of C57BL/6N.

11. A composition according to any of the claims 1 to 10, wherein the LIF is human, murine or rabbit LIF.

12. A composition for the derivation, maintenance or growth of germ line competent embryonic stem cells of mammals comprising conditioned medium of cells, said composition characterised in that it contains less than 2 ng /ml Leukemia Inhibitory Factor (LIF).

13. A composition according to claim 12, wherein the LIF is mammalian LIF such as human, murine or rabbit LIF.

14. A composition according to claim 12 or 13, characterised in that it contains less than 0,2 ng / ml Leukemia Inhibitory Factor (LIF).

15. A composition according to claim 12 or 13, characterised in that it contains less than 0,02 ng / ml Leukemia Inhibitory Factor (LIF).

16. A composition according to any of claims 12-15 characterised in that the composition is suitable for embryonic stem cells of a non-human mammal selected from the group of primates, pigs, cows, horses, goats, sheep, cats, dogs and rabbits.

17. A composition according to any of claims 12-15, characterised in that the composition is suitable for embryonic stem cells of a non-human mammal which is a rodent.

18. A composition according to claim 17 wherein the non-human mammal is Rattus sp.

19. A composition according to claim 17 wherein the non-human mammal is Mus sp.

20. A composition according to claim 19 wherein the non-human mammal is a mus musculus mouse strain of any of the strains with a genetic background selected from the group of 129/SvEv, C57BL/6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/10la, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster.

21. A composition according to claim 19 wherein the non-human mammal is selected from the group of Mus musculus strains with a genetic background of C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola,

C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster

22. A composition according to claim 19 wherein the non-human mammal is a Mus musculus strain with a genetic background of 129/SvEv or with a genetic background of C57BL/6N.

23. A composition according to any of claims 1-22 wherein the conditioned medium of cells is from an immortal fibroblast cell line.

24. A composition according to any of claims 1-12 wherein the conditioned medium of cells is from the rabbit Rab9 cell line (ATCC catalogue number CRL-1414)

25. A composition for the derivation, maintenance or growth of pluripotent embryonic stem cells of mammals for at least 10 passages as undifferentiated cells comprising conditioned medium of cells transfected with DNA encoding LIF, said composition characterised in that it is supplemented with a serum other than fetal bovine serum such as, fetal horse serum, fetal sheep serum, fetal goat serum, newborn mammal serum, adult mammal serum or serum substitute.

26. A composition according to claim 25 wherein the non-human mammal is selected from the group of non-human primates, pigs, cows, horses, goats, sheep, cats, dogs or rabbits

27. A composition according to claim 25 wherein the non-human mammal is selected from the group of rodents.

28. A composition according to claim 27 wherein the non-human mammal is Rattus sp.

29. A composition according to claim 27 wherein the non-human mammal Mus sp.

30. A composition according to claim 29 wherein the an non-human mammal is a mus musculus mouse strain of any of the strains with a genetic background background selected from the group of 129/SvEv, C57BL/6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster

31. A composition according to claim 29 wherein the non-human mammal is selected from the group of Mus musculus strains with a genetic with a genetic background of C57BL/6J-HPRT, BALB/cAnN, CBA CaOla, 129/SvJ, DBA/2N,

DBA/1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster

32. A composition for the derivation, maintenance or growth of embryonic cells of the mouse strain with genetic background 129/SvEv for at least 16 passages said embryonic cells being able to give rise to adult non-human mammals after aggregation with tetraploid embryos, comprising conditioned medium of cells transfected with a LIF gene, or a gene for a LIF variant said composition characterised in that it is supplemented with a serum other than fetal bovine serum such as, fetal horse serum, fetal sheep serum, fetal goat serum, newborn mammal serum, adult non-human mammal or serum substitute.

33. A composition for the derivation, maintenance or growth of germ line competent embryonic stem cells of a non-human mammal comprising conditioned medium of cells transfected with a LIF gene or a gene of a LIF variant, said composition characterised in that it is supplemented with a serum other than fetal bovine serum such as, fetal horse serum, fetal sheep serum, fetal goat serum, newborn mammal serum, adult mammal serum or serum substitute.

34. A composition according to claim 33 wherein the non-human mammal is selected from the group of non-human primates, pigs, cows, horses, goats, sheep, cats, dogs and rabbits.

35. A composition according to claim 33 wherein the non-human mammal is a rodent.

36. A composition according to claim 35 wherein the non-human mammal is Rattus sp.

37. A composition according to claim 35 wherein the non-human mammal is Mus sp.

38. A composition according to claim 37 wherein the non-human mammal is a Mus musculus mouse strain of any of the strains with a genetic background selected from the group of 129/SvEv, C57BU6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA 1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster.

39. A composition according to claim 37 wherein the non-human mammal is selected from the group of Mus musculus strains with a genetic background of

C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola,

C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster.

40. A composition according to claim 37 wherein the non-human mammal is a Mus musculus strain with a genetic background of 129/SvEv or with a genetic background of C57BL/6N.

41. A composition according to any of claims 25 to 40 wherein the cells being transfected are immortal fibroblasts.

42. A composition according to any of claims 25 to 40 wherein the cells being transfected are rabbit Rab9 cells.

43. A composition according to any of claims 25 to 40 wherein the cells are transfected with DNA encoding non-human mammalian LIF or a LIF variant.

44. A composition according to claim 43 wherein the cells are transfected with DNA encoding rabbit LIF or a rabbit LIF variant.

45. A composition according to claim 43 or 44 wherein the transfected DNA encoding LIF or a LIF variant is genomic DNA.

46. A composition according to any of claims 25 to 45 wherein the cell line is stably transfected.

47. A composition according to claim 25 to 46 wherein the cell line is Rab9 clone #19 which has been deposited with the BCCM under accession number LMBP 5479 CB.

48. A composition according to any of the previous claims supplemented with one or more compounds of the group selected from interleukin, oncostatin, ciliary neurotrophic factor, stem cell factor, basic fibroblast growth factor, and cardiotrophin.

49. A composition comprising conditioned medium from a Rab9 fibroblast cell line.

50. A composition comprising conditioned medium of cells transfected with DNA encoding LIF or a LIF variant, wherein the ceils being transfected are immortal fribroblast cells.

51. A composition according to claim 50 wherein the DNA encodes rabbit LIFor a rabbit LIF variant

52. A composition according to claim 50 or 51 wherein the transfected DNA encoding LIF or a LIF variant is genomic DNA.

53. A composition according to any of claims 50 to 52 wherein the cell line is stably transfected.

54. A composition according to claim 50 wherein the cell line is Rab9 clone #19 which has been deposited with the BCCM under accession number LMBP 5479

CB.

55. A method of deriving, maintaining or growing pluripotent embryonic stem cells from a non-human mammal comprising the step of cultivating the cells for at least one passage in the composition of any of claims 1 to 11 or 25 to 32.

56. Non-human mammalian pluripotent embryonic stem cells obtained by the method of claim 55.

57. A method of deriving maintaining or growing germ line competent embryonic stem cells of a non-human mammal comprising the step of cultivating the cells for at least one passage in the composition of claim 12 to 24 or 33 to 40.

58. Non-human mammalian germ line competent embryonic stem cells obtained by the method of claim 57.

59. Pluripotent embryonic stem cells from a non-human mammal selected from the group of primates, pigs, cows, horses, goats, sheep, cats, dogs rabbits and rodents such as rats and mice with the genetic background of 129/SvEv, C57BL/6N,

C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola,

C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster.

60. Pluripotent embryonic stem cells from a non-human mammal selected from the group of mice with a genetic background of C57BL/6J-HPRT, BALB/cAnN,

CBA/CaOla, 129/SvJ, DBA/2N, DBA 1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and

Swiss Webster.

61. Germ line competent embryonic stem cells from a non-human mammal selected from the group of primates, pigs, cows, horses, goats, sheep, cats, dogs rabbits and rodents such as rats and mice with the genetic background of

129/SvEv, C57BL/6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ,

DBA/2N, DBA/1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster.

62. Germ line competent embryonic stem cells from a non-human mammal selected from the group of mice with a genetic background of C57BL/6J-HPRT,

BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 CaOla, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster.

63. A method of generating transgenic non-human mammals from embryonic stem cells comprising the step of cultivating the embryonic stem cells for at least one passage in the composition of any of claims 1 to 11 or 25 to 32.

64. Transgenic non-human mammals selected from the group of primates, pigs, cows, horses, goats, sheep, cats, dogs rabbits and rodents such as rats and mice with the genetic background of 129/SvEv, C57BL/6N, C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster.

65. Transgenic non-human mammal selected from the group of mice with a genetic background of C57BL/6J-HPRT, BALB/cAnN, CBA/CaOla, 129/SvJ, DBA/2N, DBA/1 Ola, C3H/HeN, C57BL/6JOIa, FVB/N and Swiss Webster.

66. A yeast expression vector comprising a nucleotide sequence being operably linked to a promotor sequence wherein the said nucleotide sequence encodes rabbit LIF or a variant which is at least 95% identical to rabbit LIF or a functional protein fragment thereof.

67. A vector according to claim 66 wherein the nucleotide sequence encoding for rabbit LIF as shown in figure 1 [SEQ ID NO1] or figure 2 [SEQ ID NO 3].

68. A vector according to claim 67 wherein the encoded protein sequence is depicted in figure 1 [SEQ ID NO 2] or figure 2 [SEQ ID NO 4].

69. A vector according to any of claims 66 to 68 wherein the nucleotide sequence is fused in frame to a secretion signal

70. A vector according to any of claims 69 wherein the vector is pPICZα and the secretion signal is the alpha factor secretion signal.

71. A yeast cell transformed with the nucleotide sequence of any of claims 66 to 70

72. A yeast cell according to claim 71 wherein the yeast cell is a methylotrophic yeast such as Pichia pastoris

73. The yeast according to claim 72 which has been deposited with the Belgian Coordinated Collection of microorganisms under accession number MUCL-42925.

74. A method of expressing rabbit LIF or a variant which is at least 95% identical to rabbit LIF or a functional protein fragment thereof in a yeast cell according to any of claims claim 71 to 73 using recombinant DNA technology procedures.

75. A method according to 74 wherein the expressed proteins are secreted into the Pichia growth medium.

76. A composition comprising rabbit LIF or a variant which is at least 95% identical to rabbit LIF or a functional protein fragment thereof.

77. Purified rabbit LIF or a variant which is at least 95% identical to rabbit LIF or a functional protein fragment thereof.

78. A composition for the derivation maintenance and growth of non human embryonic stem cells comprising the purified rabbit LIF or a variant which is at least 95% identical to rabbit LIF or a functional protein fragment thereof.