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  • C12N5/0603—Embryonic cells ; Embryoid bodies
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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• • A—HUMAN NECESSITIES
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/235—Leukemia inhibitory factor [LIF]

Definitions

• the present invention provides a novel method for maintaining avian primordial germ cells (PGCs), in particular chicken PGCs, for prolonged periods in tissue culture.
• PGCs primordial germ cells
• These PGCs can be used for the insertion of desired DNA sequences, e.g., human genes.
• PGCs, and transgenic PGCs derived therefrom, may 0 be used to produce chimeric birds, in particular chimeric chickens.
• the modification of the genome of farm animal species is an 5 area which has been actively pursued, with varying degrees of success, for the past two decades.
• such research has been focused toward generating transgenic pigs, cows, and chickens.
• the majority of the available transgenic animals have been generated by the direct microinjection of single cell embryos with DNA constructs harboring the gene of interest.
• 0 microinjection techniques have been successful, such methods are disadvantageous in that they are costly and often suffer from low efficiency.
• ES embryonic stem
• PSCs primordial germ cells
• the first approach which comprises manipulation of the genome before lay has yielded mixed and/or inefficient results.
• the infection of oocytes in the ovary (Shuman, and Shoffner, Poultry Sci., 65:1437-1494 (1986) and pre- incubation of sperm with plasmid DNA (Gruenbaum et al., J. Cell. Biochem Supp. , 15:194 (1991) were inefficient and have not been repeated.
• the transfection of sperm cells with a plasmid construct by lipofection has been demonstrated (Squires and Drake, Anim. Biotech., 4:71-78 1993). However, germ line transmission was not reported.
• Previous PGC culturing methods have included the use of growth factor, in particular LIF or IGF-I. However, as noted, such methods have not been able to provide for prolonged culturing periods, a prevalent concern as it would facilitate the production of transgenic PGCs.
• both ES and PGC cells have been successfully used to generate chimeras by infection of such cells with replication competent and incompetent retroviral vectors.
• freshly obtained blastodermal cells have been injected into recipient embryos, resulting in birds with chimeric gonads (Carsience et al., DeveL, 117:669-675 1993)).
• Blastodermal cells can be efficiently transfected by lipofec- tion and then transferred into recipient embryos.
• germ line transmission of transfected cells has not been reported.
• An alternative route to the production of ES cells comprises PGCs.
• Procedures for the isolation and transfer of PGCs from donor to recipient embryos have been developed and have successfully generated chimeric chicken with germ line transmission of the donor genotype (Vick et al., London Ser. B, 251 :179-182 (1993), Tajima et al, Theriogenology, 40:509-519 (1993)).
• PGCs have been cryopreserved and later thawed to generate chimeric birds (Naito et al., J. Reprod. Fertil, 102:321-325 (1994)).
• this system is very labor intensive and only yields, on average, only 50 to 80 PGCs per embryo.
• PPCs primordial germ cells
• transgenic chimeric avians preferably Gallinacea and most preferably chickens
• transgenic chimeric avians for the production of heterologous protein(s) encoded by a nucleic acid sequence contained in cells introduced therein, preferably by recovery of such protein(s) from the eggs of such transgenic chimeric avians, in particular transgenic chimeric chickens.
• proteins may be obtained from the chimeric bird directly, e.g., isolated from the blood or other tissues.
• the present invention provides a novel method for maintaining avian (chicken) primordial germ cells (PGCs) in tissue culture for prolonged periods, i.e., for at least 14 days, more preferably at least 25 days, and ideally indefinitely.
• avian chicken
• primordial germ cells PLCs
• LIF leukemia inhibitory factor
• bFGF basic fibroblast growth factor
• SCF stem cell factor
• IGF-I insulin-like growth factor
• these PGCs should be useful for the production of transgenic avian PGCs, which can be used to produce transgenic chimeric avians. It is expected that these transgenic chimeric avians will be useful for recovery of heterologous proteins, which preferably can be recovered directly from the eggs of such chimeric transgenic avians. For example, such avians can be used for the production and recovery of therapeutic proteins and other polypeptides.
• the present invention obviates the problems associated with previous avian PGC culturing methods which did not enable such PGCs to be maintained in tissue culture for periods longer than about five days.
• the present inventors have surprisingly discovered that avian PGCs, preferably Gallinacea PGCs, and most preferably chicken PGCs can be maintained in tissue culture for prolonged periods, in at least 14 days, more preferably at least 25 days, and preferably longer, by the use of culture medium which contains at least the following four growth factors: leukemia inhibiting factor (LIF), stem cell factor
• the present culturing method comprises the following steps: (i) isolating PGCs from donor avian embryos; and (ii) culturing said isolated avian PGCs in a culture medium containing relative amounts of LIF, bFGF, SCF and IGF-I effective to promote their proliferation, for a prolonged time, i.e., at least 14 days, in tissue culture. Prolonged periods, as defined above, refers to a culture period 14 days or longer.
• the present inventors elected to isolate avian PGCs from chicken eggs which had been incubated for about 53 hours (stage 12-14 of embryonic development), removal of embryos therefrom, collection of embryonic blood from the dorsal aorta thereof, and transferral thereof to suitable cell culture medium (Ml 99 medium). These PGCs were then purified by ficoll density centrifugation, and resuspended in lO ⁇ l of the growth factor containing culture medium of the present invention.
• Other methods for isolating PGCs are known and may alternatively be used.
• the isolated PGCs are then counted and separated manually (e.g., using a pipette). Thereafter, PGCs collected from these different avian embryos are pooled (to increase PGC numbers) and incubated in the subject growth factor containing medium.
• This culture medium hereinafter referred to as "complete” medium contains LIF, bFGF, SCF and IGF-I as well as other substituents typically comprised in PGC and embryonic stem cell medium. More specifically, the subject "complete" medium will preferably comprise ⁇ -MEM, a well known commercially available cell growth medium to which has been added the above four growth factors and which additionally includes 10% fetal calf serum, 2 mM L-glutamine, 0.56% antibiotic/antimitotic, 34.56 mM 2- ⁇ mercaptoethanol, 1.0 U/ ⁇ l LIF, 40.0 pg/ ⁇ l bFGF, 60.0 pg/ ⁇ l IGF-I and 80.0 pg/ ⁇ l of SCF.
• ⁇ -MEM a well known commercially available cell growth medium to which has been added the above four growth factors and which additionally includes 10% fetal calf serum, 2 mM L-glutamine, 0.56% antibiotic/antimitotic, 34.56 mM 2- ⁇ mercaptoethanol
• these growth factors can be varied with PGCs being successfully maintained in tissue culture.
• the respective amounts of these growth factors may be increased with no adverse effects.
• these preferred amounts may vary, e.g., if PGCs of other avians are cultured.
• ⁇ -MEM tissue culture medium
• other media may be substituted therefor, provided that these four essential growth factors are also present.
• Applicants particularly contemplate modification of the subject "complete media" to eliminate fetal calf serum, because of its undefined and variable composition.
• feeder cells may also be useful.
• fibroblasts preferably avian fibroblasts, and most preferably Gallinacea fibroblasts (still more preferably chicken fibroblasts)
• these feeder cells may be transfected with genes encoding these growth factors, thereby eliminating the need for the exogenous addition of these factors during culturing. Essentially, the cells will provide a continual source of these growth factors.
• the amounts of these factors refer to relative amounts thereof effective to enable prolonged culturing of avian PGCs, preferably Gallinacea PGCs, and most preferably chicken or turkey PGCs, for prolonged periods in tissue culture.
• the relative amounts of these growth factors will fall within the following ranges: LIF 0.1 U/ ⁇ l to 100.0 U/ ⁇ l, more preferably 1.0 to 10.0 U/ ⁇ l and most preferably 1.0 to 2.0 U/ ⁇ l;
• these preferred ranges may vary, e.g., if ⁇ -MEM is substituted by another growth medium and if other types of avian PGCs are cultured.
• these PGCs can be maintained for long periods in culture with the successful production of chimeric avians.
• the cells have been maintained in tissue culture for up to about 4 months, with apparently no adverse effects.
• cells of up to 25 days have been tested for their ability to effectively colonize avian embryonic gonads and produce chimeric birds.
• these cells can be cultured indefinitely, with retention of the ability to produce chimeric birds.
• PGCs will be transferred into recipient avian embryos according to the methods disclosed in the example while follows. Thereafter, successful chimera production is evaluated based on migration and colonization of PGCs in the gonads, retention of PGC phenotype, or by looking for the presence of donor PGCs in gonads after hatching and breeding.
• the inventors selected genotypes which are easily followed which affect coloration. Donor birds were white broiler type and recipient birds were black feathered birds, respectively, having specific potential genotypes.
• the putative chimeras were black feathered and produced black/white progeny when mated with black birds. Thereby, successful chimeras were demonstrated based on the production of black/white feathered progeny produced after mating the putative chimeric bird with another black feathered bird.
• Bar Rock birds were used as recipients, and white feathered birds used as donors. Putative chimeric birds were demonstrated based on the production of white feathered progeny having some barred feathers.
• the subject method should be applicable for introducing any desired trait by chimerization. This will, of course, depend on the genotypic properties of the transferred PGCs.
• a significant application of the subject PGCs which can be maintained in culture for long periods, is for the production of chimeric avians.
• This will be accomplished by introducing a desired DNA sequence into the cultured PGCs.
• Means for introducing DNAs into recipient cells are known and include lipofection, transfection, microinjection, transformation, microprojectic techniques, etc.
• the present inventors initially elected to introduce a vector containing a reporter gene by lipofection.
• transiently transfected PGCs were produced, a stable transfected cell line has not, as yet, been isolated. However, it is expected that this can be accomplished by known techniques using the subject PGCs.
• a DNA will be introduced that encodes a desired gene, e.g., therapeutic polypeptide, growth factor, enzyme, etc, under the regulatory control of sequences operable in avians.
• these regulatory sequences will be of eukaryotic origin, most preferably avian, e.g., chicken regulatory sequences. Promoters operable in avian cells, e.g., derived from avian genes or viruses are known in the art.
• a stable cell line which produces the desired protein will be isolated and used for chimera production.
• the introduced DNA contain a marker DNA, the expression of which is easily detected, to more easily identify cells containing the inserted DNA.
• selectable markers include ⁇ -lactamase, ⁇ -galactosidase, neomycin phosphotranspherase, etc.
• transgenic chimeric avians Injection of the resultant transgenic PGCs into avian embryos will then result in the production of transgenic chimeric avians.
• the desired protein will then be recovered from the eggs of these transgenic avians, thereby providing a continual supply of the protein.
• the protein can be recovered from chimeric birds directly, e.g., isolated from the systemic circulatory system.
• E/E and I/I broiler type chickens have been used as donors of PGCs to develop the long term PGC culture system. Two types of bird were used as recipient embryos, a dominant black feather (El- and i/i) chicken line and a Bar Rock (E/E and i/i) line. Dominant black birds injected with white broiler (WB) type PGCs are referred as E/-(WB) and Bar Rock birds injected with white broiler type PGCs are referred as BR(WB).
• E/-(WB) and Bar Rock birds injected with white broiler type PGCs are referred as BR(WB).
• PGCs were collected from the dorsal aorta with a fine micropipette as described by Naito et al, Mol. Reprod. Dev., 37:167-171 (1994). PGCs from 20 embryos were pooled in Hanks' solution supplemented with 10% fetal bovine serum and concentrated by Ficoll density gradient centrifugation (Naito et al, Mol. Reprod. Dev., 39:153-171 1994). PGCs were counted and distributed in 10 ⁇ l drops of culture medium (DMEM, containing differing amounts of growth factors) at about 100 PGCs per drop. Culture drops were overlaid with sterile light mineral oil. Injection of PGCs into recipient embryos
• DMEM culture medium
• Stage 14-15 embryos were used as recipient embryos. After placing the egg on an appropriate surface, time was allowed for the developing embryo to position itself on the upper side of the resting egg. A small, about 10 mm “window” or less in the shell was made with a fine forceps. The embryo was brought close to the surface by adding a mixture of phosphate buffer saline with 4% antibiotics. After accommodating the embryo to visualize its heart, the marginal vein and/or dorsal aorta could be easily identified. Two hundred donor PGCs in 2 ⁇ l of media containing 0.04% trypan blue were taken into a micropipette. PGCs were injected into the dorsal aorta of the recipient embryo.
• PGCs were stained with Dil fluorescent stain. Embryos were collected after 24 hours of transfer, placed on a petri-dish and observed under an inverted microscope equipped for epi-fluorescent analysis.
• PGC culture conditions Several concentrations of human leukemia inhibitory factor (Lif), human basic fibroblast growth factor (bFGF), human insulin-like growth factor (IGF-I) and human stem cell factor (SCF) have been tested. Likewise, mitomycin treated chicken fibroblast and mouse STO cell feeder layers were tested. PGCS LONG-TERM CELL CULTURE MEDIUM
• the complete cell culture medium comprised the following substituents: ⁇ -MEM (BioWhittaker, Walkersville, MD, Cat# 12- 169F), 10% fetal calf serum (Hyclone, Logan, UT, Cat# 30070.03), 2 mM L- glutamine (Sigma, St. Louis, MO, Cat# G7513), 0.48% antibiotic/antimycotic (Signa, St.
• LIF leukemia inhibitory factor
• b-FGF basic fibroblast growth factor
• IGF-I insulin like growth factor
• SCF stem cell factor
• the final medium contains now the following growth factor concentrations: 0.0125 U/ ⁇ l of leukemia inhibitory factor (LIF), 0.5 pg/ ⁇ l of basic fibroblast growth factor (bFGF), 1.125 pg/ ⁇ l of insulin like growth factor-I (IGF-I) and 8.0 pg/ ⁇ l of stem cell factor (SCF).
• LIF leukemia inhibitory factor
• bFGF basic fibroblast growth factor
• IGF-I insulin like growth factor-I
• SCF stem cell factor
• a particularly preferred culture medium for maintaining primordial germ cell cultures will comprise the same substituents as above wherein the amounts of LIF, IGF-I, SCF and bFGF are as follows:
• PGCs were found to form large, dense, loosely adherent clumps of cells (some of the clumps have several hundreds of cells in them) within 3 to 4 days after collection. At the end of 7 days the clumps start to have large numbers of dead cells and cellular debris surrounding them. PGC clumps survive up to four weeks before they become cell monolayers. At weeks 1, 2 and 3, clumps have been dissociated, stained with a vital dye Dil and transferred into recipient embryos. At all three time-points cells were found in the gonads of some of the recipient embryos. The number of cells and the number of embryos showing stained PGCs in the gonads was inversely proportional to the age of the
• the recipient egg was positioned horizontally under a dissecting scope. A small hole was pierced into the air space of the egg to lower the internal pressure of the egg and prevent leakage. A 10 mm window was opened on the ventral surface of the egg and ⁇ 1 ml of PBS with 4% antibiotic/antimitotic was injected through the hole to bring the embryo up until it was slightly less than flush with the egg shell window. To inject the PGCs, a 30 ⁇ m pipet was beveled and then pulled using a micro forge to form a fine point with polished edges. Two hundred PGCs per embryo transfer, dissociated as described below, were picked up manually using a needle-pipette and a suction tube.
• Chicken PGCs are positive for periodic acid Schiff staining (PAS) and are claimed to be positive for alkaline phosphatase. However, there is no convincing evidence that chicken PGCs are positive for the latter.
• PAS periodic acid Schiff staining
• their phenotype was evaluated by transferring cells to recipient embryos and evaluating their presence in the gonads of the developing embryo. This method required culturing the PGCs in 100 ⁇ g/ml Dil in a ⁇ -MEM medium and rinsing prior to transfer to recipient embryos. Twenty-four hours post-transfer recipient embryos were removed and placed under an inverted microscope.
• Dil labeled cells observed in the gonads were interpreted as successful PGC migration to the gonads and confirmation of retention of PGC characteristics.
• a second method to evaluate the retention of the PGC phenotype was pursued by letting recipient embryos go to hatching and then evaluate the presence of donor PGCs in their gonads after breeding.
• the first strategy used recipient black feathered birds with possible genotype i/i, E/E, s/s, b/b and donor white feathered broiler type birds with genotype I/I, E/E, S/S, B/B.
• recipient animals were chimeric, that is to say that contain their own PGCs and donor PGCs in their gonads, they were mated to pure black feathered birds. If the resulting progeny was all black feathered then the animal was assumed to be non chimeric. However, if some of the progeny was white feathered with some black feathered patches then the recipient animal would be chimeric.
• Putative chimeric E/-(WB) birds when crossed to WB birds produced pure white chicks when they originated from a donor (WB) PGC and, white with black speckled feathers chicks when they originated from the (E/-) PGC.
• WB donor
• BR white-speckled black chicks when they originated from a (BR) PGCS.
• Crosses between putative BR chimeric birds were also done. For the latter, white chicks were produced when fertilization between two (WB) PGCs occurred and black chicks were the result of fertilization with two (BR) PGC.
• the intermediate white chick with speckled black feathers only happened when a (BR) PGC was fertilized by a (WB) PGC. LONG-TERM CULTURES BEYOND 25 DAYS
• PGC clumps form rapidly spreading monolayers. These monolayers of cell shave a flat adherent base and looser clumps and chains of PGC like cells on the upper surface. Some packets of these monolayers of cells remain PAS positive. Dil stained cells obtained from these monolayers have been transferred to recipient embryos. Some embryos have shown few cells localized in their gonads. Cell monolayers have been passaged successfully. Generally, these cells are capable of undergoing 3 to 5 passages before they start to slow down their proliferation, age and become fibroblastic looking. There are few cell lines that have gone through multiple passages and continue to thrive without apparent differentiation for about four months in continuous culture.
• Chimeric chickens were generated from fresh and cryopreserved PGCS. Twenty-five (74%) out of 34 putative chimeric chickens, produced with fresh PGCs transfers, proved to be true chimeric animals after progeny testing. Thirty (88%) out of 34 putative chimeric birds, produced with cryopreserved PGCs, were demonstrated to be true chimeric chickens. In all cases, at least 40 progeny were produced and the number of donor PGCs that were fertilized per chimeric bird varied from 1.4% to 100%, with the majority ranging between 30% to 60%. Assuming that the latter is a reflection of the number of PGCs that migrated to the gonad after injection, then the range of success per injection was varied.
• PGC phenotype under long term culture conditions After collection, PGCs are recognized by their size and by the presence of libid droplets in their cytoplasm. At about 48 hours after collection, PGCs clump together and start dividing as evidenced by the growth in size of the clump and the number of cells observed after trypsin dissociation of the clump. Only PGCs that form clumps survive, all others die. Generally, a culture starting with 100 PGCs would end up with an average of 600 to 800 PGCs within seven days. Clearly some PGCs divide, albeit not at an efficient rate. However, as indicated above, these PGCs maintain their ability to migrate to the gonads. LONG-TERM CULTURES BEYOND 25 DAYS
• PGC clumps form rapidly spreading monolayers. These monolayers of cells have a flat adherent base and looser clumps and chains of PGC like cells on the upper surface. Some packets of these monolayers of cells remain PAS positive. Dil stained cells obtained from these monolayers have been transferred to recipient embryos. Some embryos have shown few cells localized in their gonads. Cell monolayers have been passaged successfully. Generally, these cells are capable of undergoing 3 to 5 passages before they start to slow down their proliferation to age and become fibroblast-like in appearance. There are few cell lines that have gone through multiple passages and continue to thrive without apparent differentiation for about four months in continuous culture.
• P102896 and PI 10596 Two cell lines obtained from monolayers, P102896 and PI 10596, have been frozen. The former did not show apparent differentiation and was marginally positive for alkaline phosphatase while the latter showed neuronal cell morphology and was strongly positive for alkaline phosphatase. Further characterization of PGC monolayers as described here remains to be assessed for totipotency and pluripotency. In particular, it has been shown that PGCs cultured using the above four growth factors for at least 25 days can successfully colonize the gonads and produce chimeric chickens. Also, we have maintained PGC cells in culture for up to four months. These cultures still appear to comprise cells having the desired PGC phenotype. While these cells were not tested for their ability to produce chimeric birds, based on their appearance, it is expected that they should be useful therefor.
• PGC transfection Lipofection of a vector containing the green fluorescence protein reporter gene has been used for transfection of PGCS. On average 1/50 PGCs were transiently transfected, however, no stable transfected cell line has been developed yet.
• a PGC culture system should allow for transfection and selection of PGCs while maintaining the PGC ability to migrate to the gonads. Also, as disclosed in more detail in a related application (filed on even date), chicken PGCs, August 3, 1998after prolonged culturing, revert to the ES cell phenotype, as occurs with mouse PGCs (Matsui et al. Cell, 70:841-847, 1992). Therefore, injection of dispersed ES cells into recipient blastoderms should provide another means for the generation of chimeric and transgenic chickens.

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