WO2016091272A1 - Système et procédé pour l'établissement d'une culture à long terme de cellules germinales primordiales aviaires et utilisations associées - Google Patents

Système et procédé pour l'établissement d'une culture à long terme de cellules germinales primordiales aviaires et utilisations associées Download PDF

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WO2016091272A1
WO2016091272A1 PCT/EP2014/003333 EP2014003333W WO2016091272A1 WO 2016091272 A1 WO2016091272 A1 WO 2016091272A1 EP 2014003333 W EP2014003333 W EP 2014003333W WO 2016091272 A1 WO2016091272 A1 WO 2016091272A1
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primordial germ
avian
cells
culture
avian primordial
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PCT/EP2014/003333
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English (en)
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Ulrich WERNERY
Chunhai Liu
Jorg KINNE
Renate WERNERY
Ali RIDHA
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Central Veterinary Research Laboratory
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Priority to PCT/EP2014/003333 priority Critical patent/WO2016091272A1/fr
Priority to PCT/EP2015/002481 priority patent/WO2016091385A1/fr
Publication of WO2016091272A1 publication Critical patent/WO2016091272A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/0611—Primordial germ cells, e.g. embryonic germ cells [EG]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/30—Bird
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to avian biotechnology and in particular to a system and method for establishing a long-term culture of avian primordial germ cells and uses thereof.
  • the invention is particularly useful to isolate and cultivate primordial germ cells from an early stage of avian embryos.
  • the cultured avian primordial germ cells can be used for example to preserve the genetic diversity of avian species or as a targeting vehicle cell for genetic modification for disease resistance or other purposes, and to provide an interspecies or intraspecies germline chimera, e.g. for the purpose of propagation of endangered species in captive breeding programmes.
  • a Houbara breeding project was launched at the Central Veterinary Research Laboratory (CVRL), UAE, in the year 2000, with the aim of the propagation and conservation of houbara bustards via novel biotechnologies, such as avian germ cells manipulation and germline chimera.
  • Germline chimeras were produced by transferring houbara primordial germ cells (PGCs) into domestic poultry species (Wernery U, Liu C, Baskar V, Guerineche Z, Khazanehdari KA, Saleem S, Kinne J, Wernery R, Griffin DK, Chang IK.
  • Primordial germ cell- mediated chimera technology produces viable pure-line houbara bustard offspring: potential for repopulating an endangered species.
  • PLoS One 2010; 5(12):e1 5824 Donor houbara PGCs were freshly isolated from embryos, or short-term cultured in vitro, which are limited by seasonal breeding and egg availability.
  • Germ cells are the only kind of cells to pass the genetic information to the next generation. They play a crucial role in keeping the continuity of the species. The manipulation of germ cells from different stages of gametogenesis is well documented. Emerging technologies were developed to isolate, characterize, culture, cryopreserve and transplant different kinds of germ cells between individuals of the same kind, or different species (T. Ogawa, I. Dobrinski, M. R. Avarbock, and R. L. Brinster, "Xenogeneic spermatogenesis following transplantation of hamster germ cells to mouse testes," Biology of Reproduction, vol. 60, no. 2, pp. 51 5-521 , 1999; Dobrinski, M. R. Avarbock, and R. L.
  • PGCs Primordial germ cells
  • ES pluripotent embryonic stem
  • ES pluripotent embryonic stem
  • Avian primordial germ cells first appear at stage X (Eyal-Giladi H. and Kochav S. From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick.
  • the avian PGCs enter into the vascular system, passively migrate to the vicinity of the gonadal ridge, leave the blood circulation, and migrate into the gonadal strom (Kuwana T. Migration of avian primordial germ cells toward the gonadal strom. Dev Growth Diff 1 993; 35:237- 243). Unlike mammalian PGCs, which migrate between tissues before colonizing into gonad, on the way of migration from extra-gonad, avian PGCs passively migrate with blood circulation.
  • PGCs mediated-avian germline chimera technology was developed by transferring circulating PGCs, gonadal PGCs, and cryopreserved PGCs within or between a wide range of avian species, for example chicken (Tajima A., Naito M., Yasuda Y. et al. Production of germ line chimera by transfer of primordial germ cells in the domestic chicken ⁇ Callus domesticus).
  • PGCs primordial germ cells
  • injection of primordial germ cells (PGCs) into the developing avian embryo provides a promising technology, especially because these technologies allow (i) to introduce a predetermined genotype into the germline of a recipient embryo, thereby enabling the animal to pass the desired genotype on to future generations, and (ii) to target transgene integration to specific sites within the genome, which should allow high expression levels of the transgene.
  • PGCs primordial germ cells
  • EG embryonic germ cells
  • STO feeder layer a medium with the addition of LIF, bFGF, SCF (Matusui Y., Zesebo K. and Brigid L. Derived of pi uri potential Embryonic stem cell from murine primordial germ cells inculture. Cell, 1992, 70:841 -847).
  • Van de LATE cultured chicken circulating PGCs from embryonic blood into a germline competent cell line were cultured for prolonged period, showed migration capability in vivo, and gave rise to functioning gametes in the recipient testis or ovary (Van de Lizate MC, Diamond JH, Leighton PA, Mather-Love QHeyer BS, Bradshaw R, Kerchner A, Hooi LT, Gessaro TM, SwanbergSE, Delany ME and Etches RJ. Germline transmission of genetically modified primordial germ cells. Nature, 441 : 766-769. 2006). This success was confirmed later by many other publications (Macdonald J, Glover JD, Taylor L, SangHM and McGrew MJ.
  • the present inventors invented a culture system for avian PGCs, including a source of starting material. Moreover, the present inventors also found feeder cells and soluble cytokines which are favorable for the survival, proliferation and reprogramming of avian PGCs into a germline competent cell line.
  • the present invention provides a novel system to isolate and cultivate primordial germ cells from the early stage of avian embryos, in particular of endangered wild avian species, e.g. the houbara bustard. Avian PGCs expand in the invented culture system for a prolonged period, keep the migratory capability and enable the generation of germline chimera.
  • These cells can be applied to preserve the genetic diversity of avian species, e.g. the houbara bustard, or as a targeting vehicle cell for genetic modification for disease resistance or for other purposes, and to provide an interspecies or intraspecies germline chimera, e.g. for the purpose of propagation of houbara bustard in captive breeding programmes.
  • a culture PGC line from a wild endangered bird is described herein for the first time.
  • the invention thus also provides a new approach for preserving genetic diversity and provides resources for the conservation of endangered birds, e.g. the houbara bustard.
  • the present invention provides a method for establishing a long-term culture of avian primordial germ cells, wherein the primordial germ cells are derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10.
  • culture refers to cells, which are grown under controlled conditions, generally outside of their natural environment.
  • a “long-term” culture as used herein refers to a culture, which can be extended typically through multiple passages to extend the viability of the culture.
  • a long-term culture is cultured in vitro typically at least 40 days, at least 60 days, at least 80 days, at least 100 days, at least 120 days, at least 140 days, at least 150 days, at least 1 75 days, at least 200 days, at least 225 days, at least 250 days, at least 275 days, at least 300 days, at least 325 days, at least 350 days, or even longer.
  • a long-term culture is usually a culture, which is maintained at least until passage 5, or longer.
  • the "passage” number refers to the number of transfers, i.e. to the number of times that a culture is removed from a culture vessel and placed into another culture vessel. Thereby, the cells undergo a so-called passage or subculture process. By subculturing (passage) the cells are typically kept at a sufficiently low density to stimulate further growth.
  • a primary culture refers to the first culture following the isolation of cells from their natural environment, in particular from tissue. Following the first transfer, the cells are referred to as a secondary culture (or passage 1 ). After the second transfer, the cells are referred to as a tertiary culture (or passage 2), and so on.
  • a long-term culture according to the present invention is a culture maintained at least until passage 10, more preferably maintained at least until passage 20, even more preferably maintained at least until passage 30, and particularly preferably maintained at least until passage 50.
  • avian refers to any taxonomic rank, e.g. order, family, genus, species, subspecies and race, of an organism of the taxonomic class "aves”, such as, but not limited to, bustard, chicken, turkey, duck, goose, ibis, quails, pheasants, parrots, finches, hawks, crows, ostrich, emu and cassowary.
  • aves such as, but not limited to, bustard, chicken, turkey, duck, goose, ibis, quails, pheasants, parrots, finches, hawks, crows, ostrich, emu and cassowary.
  • the terms “avian”, “bird”, “aves” or “ava” as used herein are intended to have the same meaning, and will be used indistinctly.
  • preferred avian taxonomic orders are Gruiformes, Ciconii formes, and Fa Icon / ormes, as well as Galliformes and Anseriformes, whereby the taxonomic orders Gruiformes, Ciconiiformes, and Fa/con/formes are more preferred.
  • preferred birds in the present invention are endangered birds.
  • the term "endangered bird” or “endangered avian species” as used herein refers to a bird, or a species respectively, having at least the status "VU” ("vulnerable”, i.e. high risk of endangerment in the wild) in the lUCN Red List of Threatened Species.
  • the bird and/or the species has at least the status "EN” ("endangered”, i.e. high risk of extinction in the wild), more preferably at least the status "CR” ("critically endangered”, i.e. extremely high risk of extinction in the wild), and even more preferably at least the status "EW” ("extinct in the wild", i.e. known only to survive in captivity, or as a naturalized population outside its historic range) in the lUCN Red List of Threatened Species.
  • the order Gruiformes which is particularly preferred according to the present invention, includes the family Otididae (Bustards), which is particularly preferred, and the genus Chlamydotis, which is particularly preferred and which comprises two species, houbara bustard ⁇ Chlamydotis undulata) and MacQueen's bustard ⁇ Chlamydotis macqueenif), whereby in the present invention the houbara bustard ⁇ Chlamydotis undulata) is in general particularly preferred.
  • the houbara is a medium sized bustard and includes two subspecies: C. u. undulate found in arid habitats spread across northern Africa, and C. u.
  • fuertaventurae is found on the Canary Islands. In particular the subspecies fuertaventurae of the Canary Islands is highly restricted and endangered.
  • a 1 997 survey found a total population of about 500 birds. MacQueen's bustard is distributed from the east of the Sinai peninsula in furniture, Arabia, to the Caspian Sea and extending east to the Aral Sea in Mongolia. Birds from the northern populations winter further south in Pakistan (mainly in western Balochistan), and in the dry arid zone of western India. Vagrants have historically been found as far west and north as England, and as far south as northern India (R. L. Brinster and J. W.
  • the order Ciconiiformes includes the family Threskiornithidae, which is preferred and which includes the preferred subfamily Threskionithinae and the preferred genus Geronticus which comprises the preferred species Geronticus eremita, also referred to as "northern bald ibis" or “waldrapp", which is a critically endangered bird.
  • the order Fa Icon i formes includes the preferred family Falconidae, including the preferred subfamily Falconinae, which comprises the preferred genus Falco.
  • the order Anseriformes (e.g. duck, goose, swan and allies) contains about 1 50 species of birds in three families: the Anhimidae (the screamers), Anseranatidae (the Magpie-goose), and the Anatidae, which includes over 140 species of waterfowl, among them the ducks, geese, and swans. All species in the order are highly adapted for an aquatic existence at the water surface. All are web-footed for efficient swimming (although some have subsequently become mainly terrestrial). The term includes the various strains of ducks, for example Pekin duck and Muscovy duck.
  • the order Galliformes contains the chicken, turkeys, quails and pheasants. About 256 species are found worldwide. The term includes the various strains of Gallus gallus, or chickens, for example S86N, Valo, White Leghorn, Brown Leghorn,shire, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, East Lansing, Italian-Partridge-colored, Marans, Barred Rock, Cou Nu Rouge (CNR), GF30, ISA as well as strains of turkeys, pheasants, quails, and other poultry commonly bred.
  • a "culture of avian primordial germ cells" as used herein refers to a culture wherein the specific properties of the PGCs are maintained, i.e.
  • the PGC properties can be established as outlined below. This applies in particular for the long- term culture, i.e. even after culturing the cells for a prolonged period of time as described above, the cells are still PGCs as defined in the following.
  • PGC primary germ cell
  • PGC properties i.e. PGC phenotype and/or genotype
  • the cell exhibits typical PGC morphological characteristics, e.g. rich granula inside cytoplasm, larger in size (15 - 28 pm);
  • germline specific genes in particular VASA (or CVH) or a homologue thereof and Dazl or a homologue thereof, are strongly transcribed in this cell;
  • the cell strongly expresses the protein encoded by VASA (or CVH) or a homologue thereof;
  • the cell does not contribute to somatic tissues when injected into a Stage X (EG&K) or a Stage 12-1 7 (H&H) recipient embryo; and/or
  • the cells transmits the PGC genotype through the germline when injected into
  • Stage X (EG&K) or Stage 12-1 7 (H&H) embryos (Tajima et al. (1 993) Theriogenology 40, 509-519; Naito et al., (1 994) MoL Reprod. Dev., 39, 1 53-1 61 ; Naito et al., (1999) J Reprod. Fert. 1 1 7, 291 -298).
  • each single criterion out of these five criteria (1 ) to (5) may be sufficient, however, a combination of two out of the five criteria (1 ) to (5) is preferred, a combination of three out of the five criteria (1 ) to (5) is more preferred, a combination of four out of the five criteria (1 ) to (5) is even more preferred, and a combination of all five criteria (1 ) to (5) is particularly preferred to confirm the PGC properties of a cell.
  • Embryo stages are referred to herein in Roman numerals regarding the table of Eyal-Giladi and Kochav (EG&K) ((Eyal-Giladi H. and Kochav S. From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick. Developmental Biology, 1 976,49: 321 -327), whereby the EG&K table is concerned with the pre-laying stages of embryonic development.
  • Embryo stages are referred to herein in Arabic numerals regarding the table of Hamburger and Hamilton (H&H) (Hamburger & Hamilton, 1 951 , A series of normal stages in the development of chick embryo. J. Morpho!
  • stage X - XI EG&K
  • stage 1 stage 1
  • germinal crescent Swift, C.H. Origin and early history of the primordial germ cells in the chick. American Journal of Anatomy, 1 914, 1 5:483- 51 6; Ginsburg M. and Eyal-Giladi H.
  • avian PGCs separate from hypoblasts around stage 4 and become located in the lacuna between hypoblasts and hyperblasts.
  • stage 1 1 PGCs become located inside the blood vessels forming in the germinal crescent region, and begin to circulate throughout the embryonic disk in the blood (initial phase of PGC-circulation).
  • the avian PGCs enter into the vascular system around stage 1 1 , whereby mitosis of PGCs is observed throughout the PGC-circulation phase, which continues until around stage 1 6, and wherein PGCs passively migrate to the vicinity of the gonadal ridge, where they leave the blood circulation and migrate into the gonadal strom, i.e. the developing embryonic gonad, which later develops into testis or ovary (Kuwana T. Migration of avian primordial germ cells toward the gonadal strom. Dev Growth Diff 1 993; 35:237-243).
  • the earliest identification of PGCs by morphological criteria is possible approximately 8 hours after the beginning of incubation, i.e. at stage 4 (H&H).
  • the primordial germ cells reside in the germinal crescent from stage 4 (H&H) until they migrate through the vasculature during stage 12-1 7 (H&H).
  • the primordial germ cells are a small population of about 200 cells. From the vasculature, the primordial germ cells migrate into the genital ridge and are incorporated into the ovary or testes as the gonad differentiates (Swift, 1 914, Am. J. Anat. 1 5, 483 - 51 6; Meyer, (1 964) Dev Biol. 1 0,1 54-190; Fujimoto et al.
  • the primordial germ cells are derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10 (embryo stages in Arabic numerals herein are according to H&H, as described above).
  • the PGCs are located within the germinal crescent as described above.
  • the primordial germ cells are derived from germinal crescent tissue isolated from its natural environment, i.e.
  • the avian embryo at an early stage, in particular prior to the migration of the PGCs in the blood and prior to entering the developing embryonic gonad.
  • the avian embryo, from which the germinal crescent tissue is isolated may be a male or a female embryo.
  • the avian embryo is a male avian embryo.
  • the method for establishing a long-term culture of avian primordial germ cells preferably comprises a step of isolating germinal crescent tissue from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 1 0, even more preferably at stage 7 to 10.
  • the germinal crescent tissue from an avian embryo may be obtained by various methods known to the skilled person. However, it is preferred that freshly laid eggs are collected, preferably within one hour after the egg is laid, more preferably within 30 min after the egg is laid, even more preferably within 10 min after the egg is laid, and particularly preferably immediately after the egg is laid. Thereafter, preferably immediately after collecting, the eggs are incubated, e.g. in an incubator.
  • the incubation may be preferably between 35°C and 40°C, more preferably between 36°C and 39°C and even more preferably between 37°C and 38°C.
  • the incubation conditions may be chosen dependent on the species, as known by the person skilled in the art.
  • a non-limiting example for incubation conditions e.g. for eggs of houbara bustard, includes a temperature of 37.8°C, at a rocking angle of between 40° to 50°, with 25 - 45 % humidity.
  • the embryos are preferably staged, e.g. during incubation of the eggs, according to the chicken embryo staging systems described above.
  • the germinal crescent tissue is preferably dissociated to obtain a cell suspension, preferably a single cell suspension, of germinal crescent tissue cells.
  • a cell suspension can be achieved by way of different techniques known to the person skilled in the art.
  • the tissue may be dissociated by enzymatic means, e.g. Trypsin-EDTA (e.g. 0.25 % Trypsin-EDTA) may be added to the dissected germinal crescent tissue to dissociate the tissue.
  • enzymatic reaction may then be stopped by adding culture medium, e.g. PGC culture medium.
  • the tissue may be broken mechanically, e.g. by gently pipetting.
  • the resulting dissociated germinal crescent tissue may optionally be centrifuged and the cell pellets thereafter re-suspended in culture medium, e.g. PGC culture medium.
  • culture medium e.g. PGC culture medium.
  • the cell suspension may be seeded in a culture vessel, e.g. plates with wells or culture flasks, which are known to the person skilled in the art.
  • the cells of the isolated germinal crescent tissue are used to establish a primary culture.
  • the method for establishing a long-term culture of avian primordial germ cells comprises the following steps:
  • step d) transferring the primordial germ cells to a culture largely devoid of, preferably without, somatic cells derived from germinal crescent tissue.
  • cells of the germinal crescent tissue isolated in step a) are used to establish a primary culture.
  • the germinal crescent tissue is preferably dissociated, after its isolation from the embryo, in order to obtain a cell suspension, in particular a single cell suspension, of germinal crescent tissue cells. Such a cell suspension can be achieved as described above.
  • the cells are usually seeded into an appropriate culture vessel, e.g. a culture plate with wells, a culture flask etc..
  • an appropriate culture vessel e.g. a culture plate with wells, a culture flask etc.
  • the term "primary culture” (or passage 0) as used herein refers to the first culture following the isolation of cells from tissue, i.e. the first time that the cells are grown under controlled conditions, generally outside of their natural environment.
  • the cells are cultured under culture conditions as described herein, on a feeder matrix as described herein, preferably on a layer of feeder cells as described herein, e.g.
  • the medium may preferably be changed one or more times, e.g. a first change of medium at day 2, 3, or 4, preferably at day 3, followed by subsequent medium changes at every other day, wherein preferably one third to half of the medium is replaced in particular in the subsequent medium changes.
  • a first change of medium at day 2, 3, or 4 preferably at day 3
  • subsequent medium changes at every other day wherein preferably one third to half of the medium is replaced in particular in the subsequent medium changes.
  • the primary culture of cells of the germinal crescent tissue may comprise somatic cells derived from the germinal crescent tissue.
  • the somatic cells derived from the germinal crescent tissue may be isolated together with the PGCs.
  • isolated tissue samples of germinal crescent tissue may contain PGCs and somatic cells, whereby such a cell "mixture" can be used to establish a primary culture.
  • most of the somatic cells usually attach on the feeder matrix and start proliferating soon, preferably immediately, after seeding. After the primary culture, i.e.
  • the cells are transferred for the first time.
  • the first transfer of the primordial germ cells i.e. from passage 0 to passage 1 , is conducted after the PGC characteristics (e.g. PGC morphology) appeared and the somatic cells cover 65 to 95 %, preferably 70 to 90 %, more preferably 70 to 80 % of the feeder matrix.
  • This transfer method i.e.
  • transferring the PGCs when the somatic cells cover 65 to 95 %, preferably 70 to 90 %, more preferably 70 to 80 % of the feeder matrix is also preferred for the transfers subsequent to the first transfer, preferably for every transfer until passages 3 to 6, more preferably until passages 4 or 5, even more preferably in every passage in step c).
  • the method described above comprises step c).
  • step c) from passage 1 , preferably until passages 3 to 6, more preferably until passages 4 or 5, the primordial germ cells and somatic cells derived from germinal crescent tissue are co-cultured on a feeder matrix as described above.
  • a feeder matrix e.g. mouse fibroblasts, STO cells, and/or buffalo rat liver (BRL) cells, whereby BRL (buffalo rat liver) cells are preferred, in a complete culture medium as described herein.
  • BRL buffalo rat liver
  • PGCs are transferred directly after the primary culture to a culture largely devoid of, preferably without, somatic cells derived from germinal crescent tissue.
  • step c i.e. a co-culture of PGCs and somatic cells is carried out
  • the PGCs are transferred after step c), in particular directly thereafter, to a culture largely devoid of, preferably without, somatic cells derived from germinal crescent tissue.
  • the term "largely devoid of” means herein, that at least 80 %, preferably at least 85 %, more preferably at least 90 %, even more preferably at least 95 %, and particularly preferably at least 98 % of the transferred cells are PGCs, in particular showing the typical PGC morphology as described above.
  • the transfer of PGCs to a culture largely devoid of, preferably without, somatic cells derived from germinal crescent tissue is preferably carried out when the somatic cells derived from germinal crescent tissue grow slower and/or start to dilute. It is furthermore preferred that a transfer of the cells following step b) comprises the following sub-steps:
  • the term “harvesting primordial germ cells” means that as far as possible PGCs are harvested. However, due to the harvesting technique applied it may happen that a small amount of cells other than PGCs is also harvested in sub-step (i). Therefore, the term “harvesting primordial germ cells” refers to harvesting cells, wherein at least 80 %, preferably at least 85 %, more preferably at least 90 %, even more preferably at least 95 %, and particularly preferably at least 98 %, e.g.
  • the harvested cells are PGCs, in particular showing the typical PGC morphology.
  • the "harvesting the remaining cells, in particular the somatic cells derived from germinal crescent tissue” typically harvesting of the feeder cells is largely avoided (as far as possible), thus the “remaining cells” are predominantly somatic cells derived from germinal crescent tissue.
  • the “harvesting the remaining cells, in particular the somatic cells derived from germinal crescent tissue” refers to harvesting cells, wherein at least 80 %, preferably at least 85 %, more preferably at least 90 %, even more preferably at least 95 %, and particularly preferably at least 98 %, e.g.
  • the harvested cells are somatic cells derived from germinal crescent tissue.
  • a mixture of the primordial germ cells and the somatic cells derived from germinal crescent tissue is prepared, whereby the mixture preferably comprises 10 % to 50 %, more preferably 20 % to 40 %, and even more preferably 25 % to 33 %, of the cells harvested in sub-step (ii), in particular of the somatic cells derived from germinal crescent tissue, and the mixture is seeded in a vessel containing a feeder matrix.
  • a mixture of the primordial germ cells and the somatic cells derived from germinal crescent tissue usually refers to a mixture of the cells harvested in sub-step (i) and the cells harvested in sub-step (ii).
  • the mixture is a PGC-somatic cell mixed suspension.
  • the cells harvested in sub-step (i) and/or the cells harvested in sub-step (ii) and/or a mixture thereof may be examined, for example in respect to the cell morphology, e.g. by observation under a microscope.
  • step c) it is particularly preferred that, if a step c) is performed, the above described mixture is prepared and seeded in each of the transfers of the cells in step c).
  • the avian primordial germ cells are primordial germ cells from bustard, chicken, duck, quail, hawk, or ibis, e.g. northern bald ibis ("waldrapp"), preferably primordial germ cells from bustard, in particular houbara bustard, as described above. It is also preferred that the avian primordial germ cells are primordial germ cells from an endangered bird as defined above.
  • the present invention provides an isolated avian primordial germ cell, which is derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10.
  • an avian PGC is particularly useful in a culture of avian primordial germ cells and/or in an avian primordial germ cell line, in particular to obtain and/or culture a culture of avian primordial germ cells and/or an avian primordial germ cell line.
  • the present invention also provides a culture of an avian primordial germ cell as described above, i.e. wherein the primordial germ cell is derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10.
  • the term "culture” or "cell culture” as used herein refers to cells, which are grown under controlled conditions, generally outside of their natural environment.
  • the cells are grown in a culture vessel, e.g. in a well of a culture plate, in a culture flask etc..
  • the cells are grown under culture conditions as described herein.
  • the culture of avian PGCs according to the present invention preferably further comprises a feeder matrix as described herein and a culture medium as described herein.
  • This culture medium preferably comprises the growth factors FCF 2 , SCF, LIF, and IGF-1 and furthermore preferably comprises a conditioned medium as described herein, more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium.
  • the culture medium further comprises a basal medium, preferably DMEM, more preferably knockout DMEM, animal serum, preferably FBS and/or chicken serum, an antibiotic, preferably penicillin/streptomycin, and further additives, preferably including nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or ⁇ -mercapto-ethanol.
  • a basal medium preferably DMEM, more preferably knockout DMEM
  • animal serum preferably FBS and/or chicken serum
  • an antibiotic preferably penicillin/streptomycin
  • further additives preferably including nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or ⁇ -mercapto-ethanol.
  • the avian primordial germ cells are cultured in vitro for at least 5 days, at least 10 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days, whereby the longer the time period the more it is preferred.
  • the present invention also provides a cell line of an avian primordial germ cell as described above, i.e.
  • an avian primordial germ cell line wherein the primordial germ cells are derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 1 0, even more preferably at stage 7 to 10.
  • a cell line is maintained under culture conditions as described herein.
  • cell line refers in general to a colony of cells derived and developed as a subculture from a primary culture in laboratory culture.
  • a “cell line” is a cell culture selected for uniformity from a cell population derived from a usually homogeneous tissue source, whereby the avian PGC cell line according to the present invention is derived from germi nal crescent tissue as described above.
  • the cell line according to the present invention is a permanently established cell culture that will proliferate indefinitely given appropriate fresh medium and space.
  • the cell line of avian primordial germ cells according to the present invention is a cell line of primordial germ cells of a male bird.
  • the total cell number of the cell line of avian primordial germ cells according to the present invention after three months in vitro culture is more than 1 million cells, preferably more than 1 ,5 million cells, more preferably more than 2 million cells, even more preferably more than 2,5 million cells, and particularly preferably more than 3 million cells.
  • the primordial germ cell in the isolated avian primordial germ cell according to present invention, in the culture of avian primordial germ cells according to present invention, and in the cell line of avian primordial germ cells according to present invention is preferably a bustard primordial germ cell, i.e.
  • the avian primordial germ cells are primordial germ cells from an endangered bird as defined above.
  • the cell line of avian primordial germ cells according to the present invention is a cell line of primordial germ cells of a bustard, more preferably of a houbara bustard, even more preferably of a male houbara bustard.
  • An isolated avian primordial germ cell, a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention as well as the methods for establishing and maintaining a culture of PGCs according to the present invention provide an excellent system to introduce a genetic modification in avian PGCs, for example in order to introduce a genetic modification in the germ line of birds.
  • long periods in culture are typically required in order to produce a sufficient number of cells to introduce a genetic modification by conventional electroporation or lipofection protocols.
  • the avian primordial germ cell in the isolated avian primordial germ cel l according to present invention, in the culture of avian primordial germ cells according to present invention, and in the cell line of avian primordial germ cells according to present invention is preferably genetically modified.
  • DNA in particular genes
  • DNA may be inserted in the host genome, e.g. by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism.
  • DNA, in particular genes may also be removed, or "knocked out", for example using a nuclease.
  • DNA in particular genes, may also be substituted or otherwise modified.
  • Gene targeting is a further technique, wherein homologous recombination may be used to change an endogenous gene, and which can be used to delete a gene, remove exons, add a gene, or introduce point mutations.
  • a genetic modification include a reporter gene, e.g. for selecting and/or observing the PGCs, a gene related to disease resistance, a gene related to a growth hormone, a gene related to a cytokine, a gene related to an interleukin, a gene related to an interferon, a gene related to an enzyme, and/or a gene related to an immunoglobulin and/or fragments thereof.
  • the genetic modification in the isolated avian primordial germ cell, the culture of avian primordial germ cells or the avian primordial germ cell line according to the present invention, wherein the avian primordial germ cell is genetically modified is the introduction of a reporter gene or of a disease resistance into an avian primordial germ cell.
  • PGCs according to the invention may be maintained (i.e. cultured) for a long period of time in vitro prior to their introduction into recipient embryo. This long period of time allows to genetically modify said cells.
  • the cells have been in vitro cultured for at least 5 days, at least 1 0 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days.
  • a long-term culture of genetically modified PGCs according to the invention in particular a long-term culture of genetically modified PGCs obtained by the method of genetically modifying avian PGCs described herein, is also preferred according to the present invention.
  • a method of genetically modifying avian PGCs according to the present invention comprises the steps of:
  • transfected PGCs preferably by addition of a selection agent in the medium, such as for example antibiotics, amino-acids, hormones, etc.;
  • step d) culturing said genetically modified PGCs of step c) on a feeder matrix in a culture medium as previously described.
  • said culture medium of step d) is a culture medium as described herein, which comprises animal serum and preferably a combination of growth factors as described herein.
  • PGCs of step c) are genetically modified. Genetic modification may be performed, for example, by transient or stable transfection with the vector in the PGCs.
  • the PGCs are stably transfected with the vector according to techniques well known by the person skilled in the art.
  • the vector is inserted randomly into the genome of the PGCs.
  • the vector is inserted by homologous recombination into the genome of the PGCs.
  • vector refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule that can be transfected into cells and replicate independently of, or within, the host cell genome.
  • a circular double stranded plasmid can be linearized by treatment with an appropriate restriction enzyme based on the nucleotide sequence of the plasmid vector.
  • a nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together.
  • the nucleic acid molecule can be RNA or DNA.
  • plasmid refers to a small, circular DNA vector capable of independent replication within a bacterial or yeast host cell.
  • the nucleic acid vector further includes at least one regulatory sequence operably linked to a nucleotide sequence coding for a "polypeptide of interest". Regulatory sequences are well recognized in the art and may be selected to ensure good expression of the linked nucleotide sequence without undue experimentation by those skilled in the art.
  • regulatory sequences includes promoters, enhancers, and other elements that may control expression. Standard molecular biology textbooks such as Sambrook et al. eds "Molecular Cloning: A Laboratory Manual” 2nd ed. Cold Spring Harbor Press (1 989) and Lodish et al. eds., "Molecular Cell Biology,” Freeman (2000) may be consulted to design suitable expression vectors, promoters, and other expression control elements.
  • tissue-selective i.e. tissue-specific
  • promoters from which expression occurs preferentially in cells of a particular kind of tissue, compared to one or more other types of tissue.
  • An exemplary tissue- specific promoter is a chicken oviduct-specific promoter that is naturally associated with the proteins of avian egg whites including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin and the like.
  • Useful promoters also include exogenously inducible promoters.
  • promoters that can be "turned on” in response to an exogenously supplied agent or stimulus, which is generally not an endogenous metabolite or cytokine.
  • examples include an antibiotic-inducible promoter, such as a tetracycline-inducible promoter, a heat-inducible promoter, a light-inducible promoter, or a laser inducible promoter, (e.g., Halloran et al., 2000, Development 127(9): 1 953-1960 ; Gemer et al., 2000, Int. J. Hyperthermia 1 6(2): 1 71 - 81 ; Rang and Will, 2000, Nucleic Acids Res.
  • an antibiotic-inducible promoter such as a tetracycline-inducible promoter, a heat-inducible promoter, a light-inducible promoter, or a laser inducible promoter, (e.g., Halloran et al., 2000, Development 127(9)
  • polypeptide of interest or "protein of interest” refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds.
  • polypeptide includes proteins, protein fragments, protein analogues, oligopeptides, peptides and the like.
  • polypeptide contemplates polypeptides as defined above that are encoded by nucleic acids, produced through recombinant technology, isolated from an appropriate source or are synthesized.
  • Non limiting examples of polypeptides are reporter polypeptides, polypeptides relating to disease resistance, growth hormones, cytokine, interleukine, interferon, enzymes, immunoglobulins or fragments thereof.
  • the present invention also provides a method for genetically modifying an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention, wherein a transposon system, preferably a PiggyBac Transposon system, is used.
  • a transposon system preferably a PiggyBac Transposon system
  • a transposon system preferably a PiggyBac Transposon system
  • Transposon-based transgenesis is widely known to the person skilled in the art.
  • Non-limiting examples of a transposon system which may be used in the method for genetically modifying an isolated avian primordial germ cell as described above, include the Sleeping Beauty Transposon system and the PiggyBac Transposon system, whereby in the present invention the PiggyBac Transposon is preferred.
  • the PiggyBac Transposon system is well-known to those skilled in the art. Briefly, the PiggyBac Transposon system is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism. During transposition, the PB transposase, or Super PB transposase, respectively, recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and moves the contents from the original sites and efficiently integrates them into TTAA chromosomal sites.
  • ITRs inverted terminal repeat sequences
  • the powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes.
  • the TTAA- specific transposon PiggyBac is rapidly becoming a highly useful transposon for genetic engineering of a wide variety of species.
  • an isolated avian primordial germ cell according to the present invention a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention can be useful in many different ways, for example for genetic modification of the avian primordial germ cell, for obtaining a germline chimera, for preservation and/or mass production of the bird, preferably of an endangered avian species as defined above, and/or for preserving the (genetic) diversity of an avian species, in particular of an endangered avian species.
  • the present invention also provides the use of an isolated avian primordial germ cell according the invention, of a culture of avian primordial germ cells according to the present invention and/or of a cell line of avian primordial germ cells according to the present invention for genetic modification of the avian primordial germ cell, in particular as described above.
  • the genetic modification is preferably the introduction of a reporter gene or a disease resistance into avian primordial germ cell.
  • the present invention also provides the use of an isolated avian primordial germ cell according the invention, of a culture of avian primordial germ cells according to the present invention and/or of a cell line of avian primordial germ cells according to the present invention for obtaining a germline chimera.
  • Obtaining a germline chimera according to the present invention is explained in detail below, referring to a germline chimera and/or a method for obtaining a germline chimera according to the present invention.
  • the present invention also provides the use of an isolated avian primordial germ cell according the invention, of a culture of avian primordial germ cells according to the present invention and/or of a cell line of avian primordial germ cells according to the present invention for preservation and/or mass production of the bird, preferably of an endangered avian species as defined above.
  • a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention may be used for preserving the (genetic) diversity of an endangered avian species as defined above, comprising a method for establishing a long-term culture of avian primordial germ cells according the present invention and/or a method for culturing a culture of avian primordial germ cells according to the present invention as described above. Thereby, preferably a step of cryopreservation is also comprised.
  • Culture conditions and culture medium are also comprised.
  • the preferred culture conditions and the preferred culture medium which can be used in the present invention in general, in particular in the method for establishing a long-term culture of avian primordial germ cells according to the present invention, for an avian primordial germ cell according to the present invention, for a culture of an avian primordial germ cell according to the present invention, as well as for a cell line derived from an avian primordial germ cell according to the present invention are disclosed.
  • the culture conditions typically comprise a "complete culture medium".
  • the term "culture medium” in general as used herein refers to a liquid or gel designed to support the growth of cells.
  • a “complete culture medium” refers to a basal medium, preferably a basal synthetic medium, supplemented with at least one growth factor and animal serum.
  • Non- limiting examples of complete culture media are described in WO 03/076601 , WO 05/007840 , EP 787180, US 6,1 14,1 68, US 5,340,740, US 6,656,479, US 5,830,51 0 and in Pain et al. (1 996, Development 122:2339-2348).
  • basal medium refers to a medium that allows, by itself, at least cell survival, and preferably, cell growth.
  • a basal medium has a classical medium formulation.
  • Non-limiting examples of basal media include BME (Eagle's Basal Medium), MEM (minimum Eagle Medium), medium 199, DMEM (Dulbecco's modified Eagle Medium), Knockout DMEM, GMEM (Glasgow modified Eagle medium), DMEM-HamF12, Ham-F12 and Ham- F10, Iscove's Modified Dulbecco's medium, MacCoy's 5A medium, and RPMI 1640.
  • Basal media usually comprise one or more inorganic salts (for example CaCI 2 , KG, NaCl, NaHCO3, NaH 2 PO4, MgSCt, etc.), one or more amino-acids, one or more vitamins (for example thiamine, riboflavin, folic acid, D-Ca pantothenate, etc.) and/or one or more other components such as for example glucose, beta-mercapto-ethanol, and sodium pyruvate.
  • the basal medium is a synthetic medium.
  • a preferred basal medium according to the present invention is DMEM, more preferably Knockout DMEM, which is a basal medium optimized for growth of undifferentiated embryonic and induced pluripotent stem cells.
  • the basal medium is supplemented with at least one growth factor and animal serum.
  • growth factor refers to an exogenous growth factor added to the culture medium, which promotes the survival and the growth of the avian PGCs in culture.
  • Growth factors comprise in particular cytokines and trophic factors. In the present invention such cytokines, whose action is mediated through a receptor which is associated with the gp130 protein, are preferred.
  • Preferred cytokines include leukemia inhibitory factor (LIF), interleukin 1 1 (IL1 1 ), interleukin 6 (IL6), interleukin 6 receptor, Ciliary Neurotrophic factor (CNTF), oncostatin and cardiotrophin, which have a similar mode of action with a recruitment at the level of the receptor of a specific chain and the combination of the latter with the gp1 30 protein in monomeric or sometimes heterodimeric form.
  • Preferred trophic factors are Stem Cell Factor (SCF), Insulin Growth factor 1 (IGF-1 ) and Fibroblast Growth Factor (FGF), more preferably basic FGF (FGF 2 or bFGF). As growth factors, a combination of FGF 2 , SCF, LIF, and IGF-1 is particularly preferred.
  • the concentration of each of the growth factors, in particular of each of FGF 2 , SCF, LIF, and IGF-1 , in the culture medium is preferably from about 0.01 to 500 ng/ml, more preferably from about 0.1 to 100 ng/ml, even more preferably from 0.5 to 50 ng/ml, and particularly preferably from 1 to 20 ng/ml.
  • the culture medium comprises as growth factors about 5 ng/ml for SCF and about 10 ng/ml for each of FGF 2 , LIF, and IGF-1 , preferably without any further growth factors.
  • the growth factors are added to the culture medium in a final step, i.e. after the other ingredients of the culture medium are added and, preferably, after the pH value is adjusted, e.g. to 7.0, for example with NaOH, e.g. 1 N NaOH and/or with HCI.
  • the culture medium according to the present invention comprises animal serum.
  • the preferred animal serum is fetal animal serum and/or chicken serum.
  • the preferred fetal animal serum is fetal bovine serum (FBS), whereby ES cell tested FBS is in general particularly preferred.
  • FBS fetal bovine serum
  • the culture medium according to the present invention a combination of both, fetal bovine serum (preferably, ES cell tested FBS) and chicken serum, is particularly preferred.
  • the culture medium may comprise approximately from 6 to 9 % of fetal bovine serum (preferably, ES cell tested FBS) and/or from 1 to 4 % chicken serum.
  • animal serum comprising serum from other animal species e.g. horse, porcine, ungulate, etc..
  • animal serum comprising serum from other animal species (e.g. horse, porcine, ungulate, etc..) may also be used.
  • the final concentration of animal serum in the culture medium is preferably approximately from 1 to 25 %, more preferably from 5 to 20 %, even more preferably from 8% to 12 %. In a particularly preferred embodiment, the final concentration of animal serum in the culture medium is 10%.
  • the animal serum in the culture medium is a combination of fetal bovine serum (preferably, ES cell tested FBS) and chicken serum, whereby the percentage of chicken serum in total animal serum is preferably from 1 0 to 40 %, more preferably from 20 to 30 %, even more preferably from 22 to 28 %, e.g. 25%.
  • the remaining part in total animal serum is fetal bovine serum, preferably ES cell tested FBS.
  • the final culture medium contains 10 % animal serum it is particularly preferred that the final culture medium contains 7,5 % FBS (preferably, ES cell tested FBS) and 2,5 % chicken serum.
  • the culture medium preferably further comprises a conditioned medium.
  • a "conditioned medium” as used herein refers to a medium in which cells, preferably feeder cells, more preferably BRL (buffalo rat liver) cells, have been cultivated already for a period of time.
  • the percentage of conditioned medium in the culture medium is up to 80 %, more preferably up to 70 %, even more preferable up to 60 % and particularly preferably up to 55 % of the final medium volume.
  • the final culture medium contains at least 25 %, more preferably at least 33,3 %, even more preferably at least 40 %, and particularly preferably at least 45 % conditioned medium.
  • the culture medium comprises in its final volume 45 % to 55 % conditioned medium, more preferable 48 % to 52 % conditioned medium, e.g. 50 % conditioned medium.
  • the culture medium may be based on a conditioned medium, preferably BRL conditioned medium, i.e. in this case the culture medium does not comprise any "unconditioned" basal medium.
  • conditioned medium in particular BRL conditioned medium
  • a conditioned medium is usually obtained from cells cultured for a period of time in a medium.
  • the medium used for cultivating cells preferably feeder cells, more preferably BRL cells, i.e. the medium which is thereafter to become the conditioned medium, is preferably a basal medium supplemented with animal serum as described above.
  • DMEM/10% FBS and/or Knockout DMEM/10% FBS are particularly preferred.
  • the period of time, for which the medium is cultured with cells e.g. feeder cells, i.e.
  • the period of time for which the medium is "conditioned” is usually from 1 to 10 days, preferably from 1 to 4 days, more preferably from 2 or 3 days, even more preferably about 3 days. After such a period of time the conditioned medium is preferably collected and used. The culture of feeder cells is then supplied with fresh medium, which can again be collected after a period of time as described above. Before it is used, the conditioned medium is preferably treated by sterile filtration, e.g. through 0.22 pm filter, and optionally aliquoted and can be stored, preferably at -80°C.
  • a preferred conditioned medium is such a medium, which has been conditioned with the same type of feeder cells, which are preferably also used as feeder cells in the PGC culture.
  • a feeder cell conditioned medium may be obtained from a culture of feeder cells, e.g. from feeder cells cultured in basal medium supplemented with animal serum, preferably DMEM, more preferably knockout DMEM with 10% FBS.
  • the feeder cells preferably BRL cells
  • the feeder cells are at first seeded into a culture vessel with DMEM/10%FBS, after 2 to 4, preferably 3, days the medium is removed and replaced by knockout DMEM/10% FBS, which is collected after 2 to 4, preferably 3, days.
  • the collected conditioned medium is preferably accumulated, filtered, e.g. through 0.22 pm filter, optionally aliquoted and stored, preferably at -80°C.
  • the preferred feeder cells are BRL (buffalo rat liver) cells, preferably BRL-3A cells, or mouse fibroblast cells, e.g. STO fibroblasts.
  • BRL conditioned medium is particularly preferred according to the present invention.
  • BRL-3A cells are available from ATCC accession number CRL-1442.
  • feeder cells comprise cells from other mammalian species (e.g; ungulate, bovine, porcine species); or avian species (e.g. Gallinacea, chicken, turkey, duck, goose, quail, pheasant) may also be used.
  • the culture medium of the invention may preferably comprise in addition antibiotics, such as for example penicillin and streptomycin, in particular to prevent bacterial contamination.
  • antibiotics such as for example penicillin and streptomycin
  • a combination of penicillin/streptomycin is preferred.
  • the concentration of antibiotic in the culture medium is from 1 to 1 000 U/ml, more preferably from 1 0 to 500 U/ml, even more preferably from 50 to 250 U/ml, and particularly preferably about 100 U/ml.
  • a combination of penicillin/streptomycin maybe used in the following concentrations of antibiotic in the final culture medium: from 1 to 1000 U/ml penicillin and from 1 to 1000 pg/ml streptomycin, more preferably from 1 0 to 500 U/ml penicillin and from 10 to 500 pg ml streptomycin, even more preferably from 50 to 250 U/ml penicillin and from 50 to 250 streptomycin, and particularly preferably about 100 U/ml penicillin and about 100 pg/ml streptomycin.
  • culture medium of the present invention may preferably comprise further additives, e.g. nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or ⁇ -mercapto- ethanol.
  • further additives e.g. nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or ⁇ -mercapto- ethanol.
  • the further additives are typically used in concentrations according to the manufacturer.
  • the concentration in the culture medium may be for each nucleoside from 0.1 to 100 mg/l, preferably from 0.5 to 50 mg/l, more preferably from 1 to 10 mg/l.
  • "EmbryoMax" Nucleosides may be used, which contain, in the 100x stock solution 0.73 g/l Cytidine, 0.85 g/l Guanosine, 0.73 g/l Uridine, 0.8 g/l Adenosine, and 0.24 g/l Thymidine and which are thus preferably used in 1 /100 dilution in the culture medium.
  • the glutamine derivative may be for example L-glutamine or GlutaMax, whereby GlutaMax is preferred.
  • GlutaMax is an L-alanyl-L-glutamine dipeptide, which is available for example as 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl (100x stock solution).
  • the glutamine derivative is preferably used in the final culture medium in a concentration from 0.1 to 100 mM, more preferably from 0.5 to 50 mM, even more preferably from 1 to 10 mM, particularly preferably about 2 mM.
  • MEM-NEAA i.e. minimum-essential-medium-non-essential-amino-acids solution
  • MEM-NEAA i.e. minimum-essential-medium-non-essential-amino-acids solution
  • MEM-NEAA i.e. minimum-essential-medium-non-essential-amino-acids solution
  • MEM-NEAA i.e. minimum-essential-medium-non-essential-amino-acids solution
  • MEM-NEAA i.e. minimum-essential-medium-non-essential-amino-acids solution
  • HEPES phenol red as pH indicator
  • HEPES ⁇ 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a zwitterionic organic chemical buffering agent, which is widely used in cell culture, largely because it is better at maintaining physiological pH despite changes in carbon dioxide concentration (produced by cellular respiration) when compared to bicarbonate buffers, which are also commonly used in cell culture.
  • the concentration of the biological buffer, in particular HEPES, in the culture medium is preferably from 1 to 50 mM, more preferably from 5 to 30 mM, even more preferably from 8 to 12 mM, particularly preferably about 10 mM.
  • the pH of the culture medium is from 6.0 to 8.0, more preferably from 6.5 to 7.5, even more preferably from 6.8 to 7.2, and particularly preferably the pH value is adjusted to 7.0.
  • standard techniques may be used, for example NaOH, e.g. 1 N NaOH and/or HCI may be used.
  • Pyruvate is an intermediary organic acid metabolite in glycolysis and the first of the Embden Myerhoff pathway that can pass readily into or out of the cell. Thus, its addition to a cell culture medium provides both an energy source and a carbon skeleton for anabolic processes.
  • a preferred pyruvate is sodium pyruvate, which may also help to reduce fluorescent light- induced phototoxicity.
  • Pyruvate, preferably sodium pyruvate is preferably used in the culture medium in a concentration from 0.05 to 50 mM, more preferably from 0.1 to 10 mM, even more preferably from 0.5 to 5 mM, particularly preferably about 1 mM.
  • Beta-mercapto-ethanol (also referred to as 2-Mercaptoethanol, ⁇ - ⁇ or 2-ME) is assumed to act as a free radical scavenger. Beta-mercapto-ethanol is preferably used in the final culture medium in a concentration from 0.005 to 5.0 mM, more preferably from 0.01 to 1 .0 mM, even more preferably from 0.05 to 0.5 mM, particularly preferably about 0.1 mM.
  • a particularly preferred complete culture medium according to the present invention comprises: — a basal medium, preferably DMEM, more preferably knockout DMEM, as described above;
  • a conditioned medium more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium, as described above;
  • animal serum preferably FBS (preferably, ES cell tested FBS) and/or chicken serum, as described above;
  • nucleosides preferably nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or ⁇ -mercapto-ethanol, as described above.
  • Such a culture medium is particularly suitable for maintaining (i.e. culturing) avian primordial germ cells, preferably houbara bustard primordial germ cells.
  • the culture medium is preferably suitable for culturing avian primordial germ cells according to the present invention, i.e. PGCs derived from germinal crescent tissue isolated from an avian embryo at stage 4 to 1 1 , preferably at stage 4 to 10, more preferably at stage 6 to 10, even more preferably at stage 7 to 10.
  • the culture medium is preferably also suitable for maintaining avian primordial germ cells from derived from other, in particular later, embryo stages, for example chicken PGCs derived from the blood, i.e. embryo stage 12 to 1 7. Therefore, the culture medium according to the present invention can be widely used in maintaining avian primordial germ cells, and possibly additionally in maintaining other cells, preferably human/animal cells, more preferably ES cells.
  • the culture medium is particularly suitable for maintaining the avian primordial germ cells in the method for establishing a long-term culture of avian primordial germ cells according to the present invention, for maintaining an avian primordial germ cell according to the present invention, for maintaining a culture of an avian primordial germ cell according to the present invention, as well as for maintaining a cell line derived from an avian primordial germ cell according to the present invention.
  • the present invention provides a culture medium for maintaining avian primordial germ cells, preferably for maintaining houbara bustard primordial germ cells, comprising a basal medium, preferably DMEM, more preferably knockout DMEM; a conditioned medium, more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium; animal serum, preferably FBS and/or chicken serum; the growth factors FGF 2 , SCF, LIF, and IGF-1 ; preferably an antibiotic, more preferably penicillin/streptomycin; and preferably further additives, including for example nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or ⁇ -mercapto-ethanol.
  • a basal medium preferably DMEM, more preferably knockout DMEM
  • a conditioned medium more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium
  • animal serum
  • the culture medium as described above is in particular sufficient for the maintenance of avian primordial germ cells, preferably houbara bustard primordial germ cells, in culture for a10 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 100 days, at least 150 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days, whereby the longer the time period, the more preferable and particularly preferable is an infinite period, if refreshed appropriately and given the cells sufficient space.
  • the avian PGCs according to the present invention are typically cultured on a feeder matrix, preferably on a layer of feeder cells.
  • the feeder matrix provides the basement and produces favorable factors into the culture.
  • the term "feeder matrix" as used herein refers to feeder cells, which may be cultured cells and/or cell lines, and/or to extracellular matrix used as such. In particular, the feeder cells could be substituted with extra-cellular matrix, for example plus bound growth factors.
  • the feeder matrix is constructed in accordance with procedures known in the art.
  • the feeder matrix comprises feeder cells, more preferably the feeder matrix is a layer of feeder cells.
  • the feeder cells are preconditioned.
  • preconditioned means that the feeder cells are cultured in the presence of medium for a period of time prior to the depositing of cells originating from germinal crescent tissue from an avian embryo in contact with the feeder matrix, e.g. a time sufficient to initiate and establish production of auxiliary substances by the feeder matrix, for example, growth factors, nutrients or other factors.
  • a feeder matrix preferably feeder cells
  • a feeder matrix is preconditioned by culturing the feeder matrix by itself for one or more days, preferably up to five days, more preferably up to four days, even more preferably up to three days, particularly preferably two to three days, prior to the depositing of cells originating from the germinal crescent tissue from an avian embryo in contact with the feeder matrix.
  • feeder cells which can be used as feeder cells are known to the person skilled in the art.
  • cells of the fibroblast cell type may be used as feeder cells.
  • Preferred feeder cells are BRL (buffalo rat liver) cells, preferably BRL-3A cells (e.g. ATCC CRL-1442), and mouse fibroblast cells, e.g. STO fibroblasts.
  • BRL biuffalo rat liver
  • BRL-3A cells e.g. ATCC CRL-1442
  • mouse fibroblast cells e.g. STO fibroblasts.
  • feeder matrices comprise cells from other mammalian species (e.g; ungulate, bovine, porcine species); or avian species (e.g. Gallinacea, chicken, turkey, duck, goose, quail, pheasant) may also be used.
  • feeder cells may also be transfected with one or more expression vector allowing for example the constitutive expression of growth factors such as avian SCF (stem cell factor) in BRL cells.
  • avian SCF stem cell factor
  • this "feeder” produces the factor in a form which is soluble and/or attached in the plasma membrane of the cells.
  • the maintaining process comprises a step of establishing a monolayer of feeder cells.
  • Feeder cells are usually mitotically inactivated using standard techniques.
  • the feeder cells may be exposed to radiation, e.g. X or gamma radiation (e.g. 4000 Rads of gamma radiation) or may be treated with mitosis inhibitors, e.g. Mitomycin C (e.g. 10 pg/ml for 2-3 hours).
  • Procedures for mitotically inactivating cells are usually also detailed in the information typically sent with the cells at purchase, e.g. from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 201 10-2209.
  • ATCC American Type Culture Collection
  • Layers in particular monolayers, may optionally be cultured to about 80% confluency, preferably to about 90% confluency, and more preferably about 100% confluency. While configuration of the feeder cells as a layer, in particular as a monolayer, is the preferred configuration for the culture, any suitable configuration is contemplated to be within the scope of the present invention. Thus, for example, layers, mono-layers, clusters, aggregates or other associations or groupings of feeder cells are contemplated to fall within the scope of the present invention and are particularly contemplated to fall with the meaning of the term "matrix".
  • the culture conditions including the culture medium, as disclosed herein, are preferably applied to any avian primordial germ cell according to the present invention, including the cells of the isolated germinal crescent tissue, which are also preferably maintained under the above described conditions.
  • the present invention provides, in another aspect, a method for maintaining an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention, wherein the avian primordial germ cells are maintained in a culture medium on a feeder matrix, the culture medium comprising the growth factors FGF 2 , SCF, LIF, and IGF-1 and preferably a conditioned medium, more preferably feeder cell conditioned medium, even more preferably BRL conditioned medium.
  • the PGCs are preferably maintained for at least 10 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 100 days, at least 1 50 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days, whereby the longer the time period, the more preferable and particularly preferable is an infinite period, if refreshed appropriately and given the cells sufficient space.
  • the culture medium and the feeder matrix as used herein are a culture medium and a feeder matrix as described above, for example a complete culture medium as defined above, whereby the complete culture medium preferably comprises a conditioned medium, preferably B L conditioned medium as described above.
  • the concentration of each of the growth factors, in particular of each of FGF 2 , SCF, LIF, and IGF-1 , in the culture medium is comprised between about 0.01 to 1 00 ng/ml, preferably, 0.1 to 50 ng/ml, more preferably 1 to 20 ng/ml, and even more preferably about 5 ng/ml for SCF and about 10 ng/ml for each of FGF 2 , LIF, and IGF-1 .
  • the avian primordial germ cells are maintained in a preferred culture medium as described above.
  • the culture medium comprises:
  • a basal medium preferably DMEM, more preferably knockout DMEM, as described above;
  • a conditioned medium preferably feeder cell conditioned medium, more preferably BRL conditioned medium, as described above;
  • animal serum preferably FBS (preferably, ES cell tested FBS) and/or chicken serum, as described above;
  • nucleosides preferably nucleosides, a glutamine derivative, preferably GlutaMax, MEM-NEAA, a biological buffer, preferably HEPES, pyruvate (e.g. sodium pyruvate), and/or ⁇ -mercapto-ethanol, as described above.
  • the method for maintaining an isolated avian primordial germ cell according to the present invention, a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention includes a step of transferring the cells, i.e. placing the cells from one culture vessel into another culture vessel. Thereby, the cells undergo a subculture (passage) process as described above. By subculturing (passage) the cells are kept at a sufficiently low density to stimulate further growth.
  • the cells are dissociated mechanically and/or enzymatically. Mechanical dissociation can be achieved, for example, by gently pipetting the cells.
  • Enzymatic dissociation can be achieved, for example, by treatment with Trypsin-EDTA, e.g. 0.25 % Trypsin-EDTA.
  • a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention are transferred every three to nine days, more preferably every four to eight days, and even more preferably every five to seven days.
  • the culture medium in the method for maintaining an isolated avian primordial germ cell, a culture of an avian primordial germ cell, or a cell line derived from an avian primordial germ cell further comprises DMSO (dimethyl sulfoxide), in particular 10% DMSO, and that the method further comprises a step of freezing a culture of avian primordial germ cells.
  • DMSO dimethyl sulfoxide
  • Such freezing may be useful in cryopreservation, e.g. if a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention is used for preserving the (genetic) diversity of an endangered avian species as defined above.
  • the present invention provides a method for culturing a culture of avian primordial germ cells, wherein the avian primordial germ cells are maintained in a culture medium on a feeder matrix, the culture medium comprising the growth factors FGF 2 , SCF, LIF, and IGF-1 and preferably the culture medium comprises conditioned medium as described above.
  • the culture of avian primordial germ cells is obtained by a method for establishing a long-term culture of avian primordial germ cells as described above.
  • a culture of an avian primordial germ cell according to the present invention, or a cell line derived from an avian primordial germ cell according to the present invention as described above and in the method for culturing a culture of avian primordial germ cells according to the present invention as described above the avian primordial germ cells are primordial germ cells from bustard, chicken, duck, quail, hawk, or ibis, e.g. northern bald ibis ("waldrapp"), preferably primordial germ cells from bustard, in particular houbara bustard, as described above.
  • the avian primordial germ cells are primordial germ cells from an endangered bird as defined above.
  • the present invention also provides a method for molecular analysis of an avian primordial germ cell, in particular of a houbara bustard primordial germ cell, a primer pair for such molecular analysis and a respective oligonucleotide.
  • molecular analysis of cultured avian primordial germ cells provides an important tool to confirm that the cultured cells have the properties of primordial germ cells, e.g. in contrast to embryonic germ (EG) cells.
  • EG embryonic germ
  • molecular analysis thus refers to the expression profile of germ cell specific genes.
  • Such analysis is known in the art, for example from WO 2006/084035 for chicken PGCs.
  • germ cell specific genes examples include DAZL and VASA (or the chicken VASA homologue CVH oi the Drosophila gene VASA, respectively).
  • DAZL codes for "Deleted in azoospermia-like" protein, which is expressed in prenatal and postnatal germ cells of males and females, and mutations in DAZL were linked to severe spermatogenic failure and infertility in males.
  • VASA is essential for germ cell development and was first identified in Drosophila melanogaster, where VASA expression is seen in germ cells, specifically the germline stem cells (GSC's) of female ovaries and in the early stages of spermatogensis in the male testis.
  • GSC's germline stem cells
  • chicken homologue CVH is known to be a germline specific gene, which is restricted to cells within the germline of chickens and is expressed by approximately 200 cells in the germinal crescent (Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development, 127: 2741 -2750. 2000).
  • the molecular analysis according to the present invention may additionally refer to the expression profile of pluripotency related genes, since some stem cell related genes are known to be expressed in PGCs. Therefore, molecular analysis of the expression profile of germ cell specific genes and of the expression profile of pluripotency related genes may be combined.
  • pluripotency related genes include /V C (also referred to as c-Myc), the Oct4 homologues POUVand NANOG( . Lavial F et al., 2007, Development. 1 34(1 9):3549-63), AZ/ ⁇ Kruppel-like factor 4), and SOX2.
  • the present invention provides a method for molecular analysis of an avian primordial germ cell, in particular a method for molecular analysis of a Houbara bustard primordial germ cell, wherein an oligonucleotide is used, in particular as primer and/or probe, said oligonucleotide comprising or consisting of a sequence according to any of SEQ ID NO: 1 to SEQ ID NO: 6 or sequence variants thereof (cf. Table 1 ).
  • the present invention also provides said oligonucleotide, an oligonucleotide, in particular an oligonucleotide, comprising or consisting of a sequence selected from the group consisting of: a sequence according to SEQ ID NO: 1 or sequence variants thereof, a sequence according to SEQ ID NO: 2 or sequence variants thereof, a sequence according to SEQ ID NO: 3 or sequence variants thereof, a sequence according to SEQ ID NO: 4 or sequence variants thereof, a sequence according to SEQ ID NO: 5 or sequence variants thereof and a sequence according to SEQ ID NO: 6 or sequence variants thereof.
  • the present invention also provides a primer pair for molecular analysis of an avian primordial germ cell, in particular of a houbara bustard primordial germ cell, the primer pair comprising at least one oligonucleotide as defined above.
  • oligonucleotide refers to 6 to 50 nucleotides in length, preferably to 8 to 35 nucleotides in length.
  • a “sequence variant” as used herein refers to any alteration in a reference sequence, whereby a reference sequence is in particular any of the sequences according to SEQ ID NO: 1 to SEQ ID NO: 6.
  • a “sequence variant” has an altered sequence in which one or more of the nucleotides in the reference sequence is deleted, or substituted, or one or more nucleotides are inserted into the sequence of the reference nucleotide sequence. Nucleotides are referred to herein by the standard one-letter designation (A, C, G, or T).
  • Preferred sequence variants share at least 80%, preferably at least 90 %, more preferably at least 90 %, even more preferably at least 95% and particularly preferably at least 98% sequence identity compared to the reference sequence.
  • % sequence identity has to be understood as follows: Two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may then be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.
  • a sequence having a "sequence identity" of at least, for example, 95% to a reference sequence is intended to mean that the subject sequence is identical to the reference sequence (i.e. any sequence according to SEQ ID NO: 1 to SEQ ID NO: 6) except that the subject sequence may include up to five nucleotide alterations per each 100 nucleotides of the reference sequence.
  • up to 5% (5 of 100) of the nucleotides in the subject sequence may be inserted or substituted with another nucleotide or deleted.
  • the percentage to which two sequences are identical can for example be determined by using a mathematical algorithm.
  • a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin eta/. (1993), PNAS USA, 90:5873-5877.
  • Such an algorithm is integrated in the BLAST family of programs, e.g. BLAST or N BLAST program (see also Altschul et a/., 1 990, J. Mol. Biol. 21 5, 403-410 or Altschul et al.
  • the method for molecular analysis of an avian primordial germ cell in particular of a Houbara bustard primordial germ cell, according to the present invention comprises a step, wherein a nucleic acid of the avian primordial germ cell is amplified.
  • the amplification is preferably performed by polymerase chain reaction (PCR) or reverse-transciptase PCR (RT- PCR).
  • a preferred method for molecular analysis of an avian primordial germ cell, in particular of a Houbara bustard primordial germ cell, according to the present invention comprises a step, wherein a nucleic acid of the avian primordial germ cell is amplified using a primer pair selected from the group consisting of:
  • an oligonucleotide comprising or consisting of a sequence according to SEQ ID NO:
  • the present invention further provides a method for obtaining an interspecies or intraspecies germline chimera, comprising the following steps:
  • step (ii) Providing an interspecies or intraspecies germline chimera by introducing the primordial germ cells of step (i) into a recipient of the same or different avian species.
  • the recipient in step (ii) is an embryo, in particular an avian embryo.
  • the primordial germ cells of step (i) are primordial germ cells, which have been cultured in vitro.
  • germline chimera refers to an organism having germ cells, which are not genetically identical to the organisms own cells.
  • interspecies germline chimera the germ cells, which are not genetically identical to the organisms own cells, are derived from a different avian species than the species of the recipient.
  • intraspecies germline chimera in contrast, the recipient is of the same avian species as the germ cells, which are not genetically identical to the organisms own cells.
  • an "intraspecies germline chimera” includes for example a chimera having germ cells from a different subspecies, race etc. as compared to the organism's own cells.
  • the cells, which are not genetically identical to the organisms own cells contribute exclusively to the germ cells. Thus, typically no phenotypic chimera are observed.
  • an isolated avian primordial germ cell according to the present invention a culture of avian primordial germ cells according to the present invention and/or a cell line of avian primordial germ cells according to the present invention as well as the methods for establishing and maintaining a culture of PGCs according to the present invention, provide an excellent system enabling that the length of the PGC culture can be extended while the genotype and phenotype of the cells as true PGCs is preserved.
  • Such PGCs can be introduced into recipient embryos for example at a point in embryonic development when the germline competent cells are migrating to the gonad. Due to their nature as true PGCs, the introduced PGCs then contribute exclusively to the nascent population of spermatogonia or oogonia (i.e., the precursors of sperm and eggs) in the resulting animals, i.e. the "germline chimera". In such a resulting animal, the entirety of the somatic tissue is thus derived from the recipient embryo, whereas the germline contains contributions from the introduced PGCs, i.e. from the "donor cells", typically in addition to germline cells of the recipient embryo. If both, the introduced PGCs and the recipient embryo's cells, contribute to the germline, the offspring of such germline chimeras is derived either from the donor cell, i.e. the introduced PGC, or from the recipient embryo.
  • the primordial germ cells of step (i) are cultured in vitro iox at least 5 days, at least 1 0 days, at least 14 days, at least 25 days, at least 50 days at least 75 days, at least 1 00 days, at least 1 50 days, at least 200 days, at least 250 days, at least 300 days, and/or at least 350 days or even longer, before they are introduced into a recipient in step (ii).
  • At least 200 cultured primordial germ cells of step (i), at least 500 cultured primordial germ cells of step (i), at least 1000 cultured primordial germ cells of step (i), at least 2000 cultured primordial germ cells of step (i), at least 4000 cultured primordial germ cells of step (i), at least 5000 cultured primordial germ cells of step (i) are introduced into a recipient in step (ii).
  • the cultured primordial germ cells introduced into the recipient avian embryo may be a mixed population of female and male primordial germ cells.
  • the method for obtaining a germline chimera according to the present invention may comprise the additional step of determining the sex of recipient embryo prior the introduction of the cultured primordial germ cells.
  • the recipient and/or the cultured primordial germ cells are previously sexed before introduction.
  • female cultured primordial germ cells are introduced into a female recipient and male cultured primordial germ cells are introduced into a male recipient.
  • the cultured primordial germ cells of step (i) are genetically modified, in particular as described above.
  • a genetic modification relating to a reporter gene e.g. GFP (green fluorescent protein), and/or to a gene relating to a disease resistance may be particularly useful in the cultured primordial germ cells of step (i) in the method for obtaining a germline chimera according to the present invention.
  • a reporter gene may be used for selecting embryos, which are germline chimeras, and observing the development of the introduced PGCs.
  • the primordial germ cells of step (i) are introduced into the sub-germinal cavity of the recipient avian embryo or in the dorsal aorta of the recipient avian embryo, whereby the introduction of the cultured primordial germ cells of step (i) into the sub-germinal cavity of the recipient avian embryo is more preferred.
  • the term "subgerminal cavity” refers to the space between the blastoderm and the yolk. This space is created when the blastoderm cells absorb fluid from the albumin and secrete it between themselves and the yolk.
  • the cultured primordial germ cells of step (i) are introduced into a recipient avian embryo at a stage from stage X (EG&K) to stage 1 7 (H&H). More preferably, the recipient avian embryo is at stage X or at a stage from stage 14 to stage 1 7, even more preferably at a stage from stage 1 5 to stage 1 6.
  • stage X introduction into the sub-germinal cavity of the recipient avian embryo is preferred, whereas introduction into the dorsal aorta of the recipient avian embryo is preferred, when introduced at a stage from stage 1 4 to stage 1 7, more preferably at a stage from stage 1 5 to stage 1 6.
  • the recipient embryo may derive from a freshly laid un-incubated egg or from an incubated embryo.
  • the recipient embryo derives from a freshly laid un- incubated egg and comprises between around 5000 to around 70000 cells.
  • the donor of the avian primordial germ cells which are cultured in step (i) of the method for obtaining a germline chimera according to the present invention may be any bird. However, preferably the donor of the avian primordial germ cells is an endangered bird as defined above. Alternatively, or additionally, it is preferred that the donor of the avian primordial germ cells is of the order Gruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis, and even more preferably the donor of the avian primordial germ cells is a houbara bustard ⁇ Chlamydotis undulata).
  • the donor of the avian primordial germ cells is of the order Gruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis, and even more preferably the donor of the avian primordial germ cells is a houbara bustard ⁇ Chlamydot
  • the recipient of the avian primordial germ cells, which are cultured in step (i) of the method for obtaining a germline chimera according to the present invention may be any bird.
  • the recipient of the avian primordial germ cells is a chicken or a bird of the order Gruiformes. If the recipient of the avian primordial germ cells is a chicken, a chicken of White Leghorn strain is particularly preferred. If the recipient of the avian primordial germ cells is of the order Gruiformes, the family Otididae (Bustards) is preferred, genus Chlamydotis is more preferred and a houbara bustard (Chlamydotis undulata) is even more preferred.
  • the present invention also provides a germline chimera, having a germline comprising a primordial germ cell derived from a culture of avian PGCs according to the present invention or from an avian primordial germ cell line according to the present invention.
  • a germline chimera according to the present invention the primordial germ cells derived from a culture according to the present invention or from an avian primordial germ cell line according to the present invention are typically located in the gonads.
  • the germline chimera according to the present invention comprises a primordial germ cell as defined above, which is derived from an endangered bird as defined above, and/or from a bird of the order Gruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis, and even more preferably the primordial germ cell as defined above is derived from a houbara bustard ⁇ Chlamydotis undulata).
  • the germline chimera according to the present invention is obtainable, preferably is obtained by, a method for obtaining a germline chimera according to the present invention as described above, in particular also regarding the preferred embodiments of this method as outlined above.
  • the present invention also provides a method for obtaining a pure species offspring from an interspecies germline chimera comprising the following steps:
  • step (i) a. a pure species bird, which is of the same avian species as the donor of the primordial germ cells introduced in the interspecies germline chimera in step (i); or
  • step (iii) selecting pure species offspring, which is preferably of the same avian species as the donor of the primordial germ cells introduced in the interspecies germline chimera in step (i).
  • a contextpure species offspring refers to an offspring, which has cells of only one single avian species.
  • the offspring of an interspecies germline chimera, which is to be obtained in this method, in particular which is selected in step (iii) is not an interspecies chimera.
  • step (ii) preferably fertile birds of opposite sex are used.
  • the interspecies germline chimera obtained in step (i) may be either crossed with a pure species bird, which is of the same avian species as the donor of the primordial germ cells introduced in the interspecies germline chimera in step (i).
  • the pure species bird is a wild-type bird.
  • the resulting interspecies germline chimera is preferably crossed with a pure species houbara bustard, which is preferably wild-type.
  • step (ii) the interspecies germline chimera obtained in step (i) may also be crossed with an interspecies germline chimera obtained by a method for obtaining an interspecies germline chimera as described herein, wherein the species of the donor of the introduced avian primordial germ cells and the species of the recipient embryo correspond to the species of the donor of the introduced avian primordial germ cells and the species of the recipient embryo of the interspecies germline chimera obtained in step (i).
  • the donor of the primordial germ cells is a houbara bustard and the recipient is a chicken
  • such an interspecies germline chimera may be crossed with an interspecies germline chimera, wherein also the donor of the primordial germ cells is a houbara bustard and the recipient is a chicken.
  • the offspring selected in step (iii) is preferably F1 progeny to the interspecies germline chimera obtained in step (i).
  • the pure species offspring selected in step (iii) is of the same avian species as the donor of the primordial germ cells introduced in the interspecies germline chimera in step (i), e.g. if the introduced primordial germ cells are houbara bustard, the pure species offspring selected in step (iii) is preferably also houbara bustard.
  • step ii) a. a more detailed description of cross-breeding an interspecies germline chimera with a pure species bird, which is of the same avian species as the donor of the primordial germ cells, (step ii) a.) and of selecting pure species offspring is provided in Wernery U. et al., 2010, PLoS One. 2010 Dec 29;5(12):e1 5824, which is hereby incorporated by reference, in particular regarding the molecular analysis of chimeric embryos and of semen samples from chimeric adults, the progeny test and the species identification and parentage test of the resulting progenies.
  • the pure species offspring is an endangered bird and/or a bird of the order Gruiformes, preferably of the family Otididae (Bustards), more preferably of the genus Chlamydotis and even more preferably a houbara bustard ⁇ Chlamydotis undu/ate).
  • Figure 1 shows for Example 3 the collection of germinal crescent (circled area) containing PGCs from Houbara Bustard embryo (stage 10).
  • Figure 2 shows for Example 4 primary cultures of Houbara Bustard germinal crescent cells.
  • A Freshly seeded Houbara Bustard germinal crescent cells.
  • B Houbara PGCs appeared after three to four days culture.
  • C Single PGCs after seven days culture.
  • D Cluster of Houbara Bustard PGCs after seven days culture. shows for Example 5 derivation cultures of Houbara Bustard germinal crescent PGCs.
  • A Houbara PGCs in single cell or chain form losing granules after 10 days culture.
  • B Houbara PGCs clumps attached and losing granules after 10 days culture.
  • Example 8 shows for Example 8 the karyotype of cultured Houbara Bustards PGCs. shows for Example 9 the detection of the gene expression profile of long-term cultured Houbara Bustard PGCs. shows for Example 10 immunofluorescence stain of Houbara PGCs with anti- CVH.
  • A negative control chicken PGCs.
  • B negative control Houbara PGCs.
  • C positive control chicken PGCs with anti-CVH.
  • D positively stained Houbara PGCs with anti-CVH. shows for Example 1 1 transfection of cultured Houbara PGCs with Piggybac transposon system carrying GFP gene.
  • A Houbara PGCs grow in culture in light field.
  • Table 2 shows the formula of an example of a PGCs culture medium of the present invention, which is useful in particular for culturing Houbara Bustard PGCs.
  • Table 2 Formula of an example of PGCs culture medium
  • FGF 2 25ug/ml (2500x) 20 ⁇ 10ng/ml
  • Table 3 shows the commercially available ingredients of the above PGCs culture medium with examples of the respective suppliers.
  • Table 3 Ingredients of the example for a PGCs culture medium with examples of suppliers
  • Knockout DMEM was added to make the total volume to be 50ml. This medium can be stored at 4°C for a few weeks.
  • BRL-3A cells (Buffalo Rat Liver cells, purchased from ATCC, ATCC-Number: CRL-1442) were used to prepare a conditioned medium as follows.
  • BRL-3a cells were seeded into a T75 flask with a density of 1 .1 x 10 6 cells in 1 5-20ml DMEM (1 0% FBS).
  • the medium was conditioned for 3 days, collected, and replaced with fresh medium, for 4 times.
  • Conditioned media were accumulated, filtered through 0.22pm filter, aliquot, and stored in -80C freezer. Condition medium can be used within one year.
  • BRL-3A feeder cells were purchased from ATCC, and cultured in DMEM/10% FBS, and passaged every three days. The cells for feeder preparation are not cultured beyond one month of total culture period, before being mitotically inactivated by X-ray irradiation or Mitomycin C treatment.
  • BRL-3a cells were maintained in T75 flask in DMEM/10% FBS, and grew into about
  • BRL-3A cells were cultured into 80-90% confluent in growth media in T75 flask.
  • the medium was removed, and replaced with 15ml fresh culture medium added with Mitomycin C (Sigma m0503) at a concentration of 10pg/ml.
  • Mitotically inactivated BRL-3A cells were thawed in a 37.5°C water bath, and an equal volume of DMEM/10%FBS was added, and centrifuged at 1450rpm/ min, 4°C. The cell pellets were re-suspended in certain volume of DMEM/10% FBS. Cells were seeded in a 48 well plate (Falcon, 3230) 1 x10 5 cells/ well, or a 24 well plate, 2x10 5 /well. Feeder cells were cultured for two to three days before seeding the PGCs, in particular the Houbara Bustard PGCs. Usually, the feeder cells were generally cultured in DMEM/10% FBS until seeding of the PGCs. From seeding of PGCs onwards usually PGC culture medium as described herein is used as medium.
  • Example 3 Collection of tissue from germinal crescent of a Houbara Bustard embryo
  • Houbara Bustard eggs were obtained from the birds which were bred in captivity over three generations in a captive breeding farm, from the third generation of parent flocks. Freshly laid Houbara Bustard egg were collected, and disinfected in F10 for 20 minutes, and incubated with the blunt end up in the incubator under 37.8°C, at a rocking angle of ⁇ 45° , with 30- 40% humidity.
  • the Houbara eggs were incubated for 40-48 hours. Eggs were opened from the blunt end, and a piece of egg shell was removed by forceps. A small piece of shell inner membrane was dissected and the embryo was made visible. The embryos were observed and staged parallel to chicken embryo staging system (Hamburger V, Hamilton HL. A series of normal stage in the development of the chick embryo. J Morphol 1 951 ; 88:49-92). The embryos at stage 7 (1 somite) to stage 1 0 (1 0 somites) were used for germinal crescent collection. Any embryo younger than stage 7 was sealed and put back into the incubator until it reached the desired stage.
  • a ring prepared from filter paper with a diameter of 2 centimetres was put around the peripheral area opaca.
  • the whole embryo was dissected out with scissors along the outer edge of the paper ring ( Figure 1 ).
  • the whole embryo was taken out from the yolk, and put into PBS(-) with the abdomen side up in a petri dish.
  • Yolk granular was cleaned up by gentle pipetting.
  • the whole germinal crescent area was dissected out with the tip of a 1 ml insulin syringe.
  • Dissected tissues were transferred into 1 .5 ml Eppendorf tubes with 20 ⁇ PBS(-). Remaining tissues were kept in -20°C freezer for molecular sexing use.
  • Trypsin- EDTA 50 ⁇ 0.25% Trypsin- EDTA were added to dissociate the tissue, kept at room temperature for 2 minutes, and stopped by adding 100 ⁇ ! PGC growth media, and gently pipetted to break the tissue. The mixture was centrifuged at 1450 rpm for 5 minutes at 4°C. Cell pellets were re-suspended in 300 ⁇ PGCs culture medium, and seeded into 48 well plates on the preconditioned BRL feeder cells.
  • Example 4 Primary and derivation culture of Houbara PGCs.
  • the fibroblast like somatic cells derived from the Houbara germinal crescent grew very fast, and Houbara PGCs continued proliferating. Once they covered 80-90% of the feeders, the first passage was conducted.
  • the PGCs cells suspension was transferred into a 1 .5ml Eppendorf tube, and 1 50 ⁇ PBS (-) was added into the original well to rinse once.
  • PGCs-somatic cells mixed suspension and the remaining stromal cells were seeded individually into in two different wells with feeder cells.
  • the PGCs and stromal cells were co-cultured on feeder cells for further four to five days until the stromal cells reached to confluent again. The next passages were conducted in the same process.
  • Example 6 Maintenance culture of Houbara Bustard PGCs Established Houbara PGCs were routinely maintained in PGCs culture medium on mitotically inactivated BRL feeders. When confluent, cells can be dissociated by mechanically pipetting or enzymatically treated with Trypsin-EDTA, and passaged every five to seven days as following: The culture medium was removed, and the remaining medium was washed off with 100 ⁇ PBS(-). 50 ⁇ 0.25% Trypsin-EDTA was added, and incubated at room temperature for 30-50 seconds, and observed under the microscope, until PGCs inside the clump rounded up, and the border between cells was clearly visible. 1 0 ⁇ culture medium was immediately added to stop digestion.
  • the culture was gentle pipetted with a blue tip from the side of the well, to detach and break the PGCs clumps from the feeder layer.
  • the cell suspension was harvested and centrifuged at 1450 rpm for five minutes at 4°C, and the pellet was re-suspended with fresh medium. Mechanically, the cultures were simply pipetted with the yellow tip, and observed under the microscope, until all the PGCs clumps were detached and broke into single cells or three to five cell small clumps.
  • the cell suspension was harvested and centrifuged at 1450 rpm for five minutes at 4°C, and the pellet was re-suspended in fresh medium.
  • Cells were counted using a hemocytometer, and adjusted into a concentration of 3-5 x1 0 5 cells/ml. 500 ⁇ cell suspension per well were seeded in a 24 well plate. The cell number doubling time of Houbara PGCs in culture were calculated from five continuous passages. Cells were maintained or frozen down with freezing medium (growth media with 10% DMSO).
  • FIG. 5A Blood source PGCs with typical PGCs morphology were observed.
  • Cells started to divide immediately after seeding. After four days culture, most blood PGCs were in doublet or two to three cells clumps, or three to five cells chain like structure (Figure 5 B). However, most of blood source PGCs started losing granules and stopped dividing from days five to seven ( Figure 5 C, D). During the next two weeks culture, blood cells gradually died off. However, there were no typical PGCs observed any more from all the 10 cultures (Figure 5 E, F).
  • germinal crescent PGCs In contrast, in germinal crescent PGCs cultures, cells behaved as described in Example 5. Even though some PGCs lost their PGCs morphology, clumped, and attached, some typical PGCs could still be seen, divided in single cells form or in clump rich in granular in cytoplasm. These cells are promising to grow continuously. Thus, germinal crescent source PGCs showed better potential than migratory PGCs from embryonic circulation for derivation of a long-term culture and/or a PGCs line in vitro.
  • Example 8 Karyotype of long-term cultured Houbara Bustard PGCs To monitor the cytogenetic karyotype of Houbara PGCs after long-term culture, karyotyping of culture PGCs was conducted by preparing chromosome metaphase spreads. Briefly, Houbara PGCs were cultured for over eight months in the present system. Karyomax colcemid (Gibco 1 5210-040) was added at a final concentration of 0.1 pg/ml to PGCs culture in a 24 well plate, and incubated for one hour. After the incubation, the medium was removed, and the cells were washed with PBS(-).
  • Karyomax colcemid Gibco 1 5210-040
  • Houbara PGCs single cell suspension was harvested with Trypsin-EDTA disassociation. The cell suspension was spun down. Supernatant was removed, and the PGCs pellet was carefully mixed in the remaining solution. Hypotonic solution (0.56 % KCI) was added slowly (drop wise) to each sample and the tube was gently tapped to mix the contents. Cells were incubated in a 37°C waterbath for eight minutes, then centrifuged and gently re-suspended in the remaining supernatant. Fixative solution (75 % Methanol, 25% Acetic Acid) was added to each sample while mixing. Cells were incubated on ice for 20 minutes then washed twice with fixative by centrifugation. 20 ⁇ of the cells/fixative were dropped onto each slide.
  • Fixative solution 75 % Methanol, 25% Acetic Acid
  • Example 9 Molecular analysis of the expression profile of pluripotency related and germ cells specific genes in cultured Houbara Bustard PGCs
  • PGCs are unipotent stem cells. Some stem cells related genes were found expressed in the in vivo and in vitro cultured chicken or mammalian PGCs. When being cultured in vitro, mammalian PGCs kept proliferating and transformed into embryonic germ cells, which were pluripotent and lost their germ cell identity. In the present example, Houbara Bustard PGCs were cultured over the long-term, and still kept the PGCs morphological characteristics. The expression of stem cells related and germ cells specific genes were analyzed by means of RT- PCR (Reverse transcriptase PCR), since the Houbara genomic sequence is not available yet.
  • RT- PCR Reverse transcriptase PCR
  • the primers for detecting the expression of chicken cPOUV, cNANOG, cKLF4, cSOX2 genes were applied.
  • Primers to detect the expression of Dazl, Myc, Vasa homolog genes were designed on the basis of the falcon gene sequence (http://www.ncbi.nlm.nih.gov/). Primer sequences are shown below in Table 4:
  • RNA isolation was performed using the RNeasy mini kit (Qiagen) with the aid of the DNase digestion kit to eliminate DNA contamination. All the process followed the instructions. Extracted RNA sample was stored at -80°C until further processing.
  • RNA was obtained using RT Ipsogen kit (Qiagen). ⁇ g of RNA (10 ⁇ ) was incubated at 65°C for 5 minutes. The sample was cooled immediately on ice for 5 minutes; then centrifuged briefly (10 seconds at 1000 rpm) and kept on ice. Reverse transcription premix was prepared as following: for a total volume of 1 5 ⁇ , 5.0 ⁇ reverse transcription buffer (5x), 2.0 ⁇ dNTP 10 mM each, 5.25 ⁇ random nanomer 100 ⁇ , 0.5 ⁇ RNase inhibitor 40 U/ ⁇ , 1 .0 ⁇ reverse transcriptase 200 U/ ⁇ and 1 .25 ⁇ DTT. 1 5 ⁇ of the premix was added to the RNA sample. The sample was run on a thermal cycler using the following program:
  • PCR was performed following the Taq PCR master mix kits manual (Qiagen). A total of 25 ⁇ reaction mix per sample was prepared: 12.5 ⁇ master mix, 1 .25 ⁇ primer forward, 1 .25 ⁇ primer reverse, 8 ⁇ H 2 0, 2 ⁇ Houbara Bustard PGCs cDNA. The reaction complete mix was then quickly pipetted to mix, centrifuged and placed in the thermal cycler.
  • PCR (with 40 cycles) was run at 95°C for 20 minutes, 95°C for 30 seconds, 55 - 59°C for 30 seconds (annealing temperature 55°C for amplification of Dazl, Myc, Vasa, cPOUV, cNANOG, cKLF4, and cSOX2), 72°C for 1 minute, and 60°C for 30 minutes.
  • PCR products were loaded on 1 % agarose gel in TBE buffer and run for 40 minutes at 60 V. To confirm the specificity of amplification, PCR product was sequenced and compared with the homologue genes of other species using BLAST.
  • pluripotent related genes Pouv, Nanog, Klf4, Sox2,Myc
  • germ cells related homolog genes Dazl and Vasa
  • VASA is an RNA binding protein with an RNA dependent helicase.
  • the vasa gene is essential for germ cell development and was first identified in Drosophila melanogaster. It is homologous to a DEAD (Asp-Glu-Ala-Asp)-family protein in the mouse.
  • DEAD Adethyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-phenyl-N-N-N-N-N-N-N-N-N-N-N-binding of oskar mRNA.
  • Chicken vasa homolog (CVH) gene was identified in 2000 (Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development, 127: 2741 -2750. 2000), and expressed in PGCs and adult germ cells. Anti-CVH
  • Houbara PGCs were cultured, and single cell suspension was harvested.
  • Chicken PGCs were cultured under conditions as published (Van de Lked MC, Diamond JH, Leighton PA, Mather-Love C,Heyer BS, Bradshaw R, Kerch ner A, Hooi LT, Gessaro TM, SwanbergSE, Delany ME and Etches RJ. Germline transmission of genetically modified primordial germ cells. Nature, 441 : 766-769. 2006), which is incorporated by reference herein, and harvested as positive control. Cells were fixed in 4 % PFA for 15 minutes. Smears were prepared on TESPA coated slides.
  • Example 1 1 Transfection of cultured Houbara PGCs with Piggybac transposon system carrying GFP gene.
  • exogenous GFP gene was introduced into cultured Houbara PGCs by Piggybac transposon system (Macdonald J, Taylor L, Sherman A, Kawakami K, Takahashi Y, Sang HM and McGrew MJ. Efficient genetic modification and germ-line transmission of primordial germ cells using piggy-Bac and Tol2 transposons. Proceedings of the National Academyof Sciences of the USA, 109: 1466-1472. 2012).
  • Cells were transfected by electroporation using pipette-type electroporator (Invitrogen).
  • Houbara PGCs were cultured as described above. Media were removed carefully and PGCs, which slightly attached to the feeder layer, were rinsed with PBS (-). Cells were harvested after dissociated with Trypsin EDTA. PGCs single cell suspension was adjusted into a concentration of 5x1 OYrnl with growth media, and kept at room temperature.
  • Freshly laid White Leghorn chicken eggs were used as the recipient.
  • a window with a diameter of 1 cm was made by removing piece of eggshell from the blunt end.
  • the inner shell membrane was removed to expose the blastoderm.
  • 5000 Houbara PGCs in 1 ⁇ of culture medium were injected into the subgerminal cavity. Windowed eggs were fully filled with albumin, sealed with cling wrap and tightly covered with a cell culture dish. Eggs were put back into the incubator with the sharp end up. After 20 hours of incubation, the eggs were opened. The whole embryo and peripheral tissue was dissected, and observed under fluorescence microscope to check the distribution of Houbara PGCs. The remaining eggs were opened on day three.
  • Houbara PGCs clumped together, and some of clumps attached on the feeder cells, though no morphological EG cells colonies were observed. 25 embryos developed beyond 14 days with fully feather development, no phenotypic chimera were observed. Injection of Houbara Bustard PGCs into blood circulation of chicken embryo (stage 15-16) Fertilized White Leghorn chicken embryos were incubated to stage 1 5-1 6 for 58 hours at 37.8°C, 60 % relative humidity. To expose the embryo, a small window was opened by removing pieces of eggshell from the sharp end. A number of 5000 Houbara PGCs was injected into the dorsal aorta of chicken embryos with a fine glass needle.
  • Injected eggs were sealed with double layer of parafilm. Eggs were returned to the incubator, and continued incubation for 5 days till stage 33. Gonadal tissue were dissected and observed under inverted fluorescence microscope, then mechanically and enzymatically dissociated into single cell suspension. The number of green Houbara PGCs was counted. A total number of 30 eggs were injected. 23 embryos survived on day 8. Gonadal tissues were dissected from 15 embryos.
  • Freshly laid Houbara eggs were incubated for 80-85 hours with the blunt end up at 37.8°C in a dry incubator. Eggs were opened from the sharp end to expose embryos. A number of 5x10 3 Houbara green PGCs (HB513g, male) with a total culture period of over 1 0 months) in 1 ⁇ growth media were injected into the recipient Houbara embryos (stage 15-1 6, H&H). Injected eggs were sealed with double layers of parafilm, and returned back to the incubator to hatch. Eggs were candled daily after 14 days incubation. Gonadal tissue were dissected from the dead embryos, and observed under fluorescence microscope.
  • Houbara Bustard PGCs derived germ cells were found from five gonadal samples (male in Figure 1 1 , female in Figure 12). In the testis, most donor green cells were found in the left gonad, and in the ovary tissue, cells were found in the cortex area. The present results suggested that Houbara Bustard PGCs could still keep the capability to migrate and proliferate in recipient gonad, even after long-term in vitro culture.

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Abstract

La présente invention concerne un système et un procédé permettant d'établir une culture à long terme de cellules germinales primordiales aviaires et les utilisations associées L'invention est particulièrement utile pour isoler et cultiver des cellules germinales primordiales à partir d'un stade précoce d'embryons aviaires. En particulier, la présente invention concerne un procédé permettant d'établir une culture à long terme de cellules germinales primordiales aviaires, les cellules germinales primordiales étant dérivées de tissu croissant germinal isolé à partir d'un embryon aviaire au stade 4 à 11, de préférence au stade 4 à 10, de préférence encore au stade 6 à 10, de préférence encore au stade 7 à 10, En conséquence, la présente invention concerne également une culture cellulaire et une lignée cellulaire correspondante de cellules germinales primordiales aviaires. Les cellules germinales primordiales aviaires cultivées peuvent être utilisées, par exemple, afin de préserver la diversité génétique d'espèces aviaires ou comme cellule vecteur de ciblage aux fins de modification génétique, pour la résistance aux maladies ou à d'autres fins, afin de fournir une chimère à lignée germinale inter-espèces ou intra-espèce, par exemple dans le but de propagation d'espèce en voie de disparition dans des programmes de reproduction en captivité. Ainsi, les cellules germinales primordiales aviaires peuvent être génétiquement modifiées. En outre, la présente invention concerne également un procédé permettant de maintenir une culture de cellules germinales primordiales aviaires, ainsi qu'une chimère à lignée germinale et un procédé pour obtenir une chimère à lignée germinale.
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CN116286612A (zh) * 2023-03-30 2023-06-23 广东省农业科学院动物科学研究所 以mTOR、SMAD信号通路和p53基因为靶点在提高鸡PGCs体外建系效率中的应用

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CN111500531A (zh) * 2020-05-15 2020-08-07 扬州大学 一种鸡PGCs在体外长期培养的方法
CN116286612A (zh) * 2023-03-30 2023-06-23 广东省农业科学院动物科学研究所 以mTOR、SMAD信号通路和p53基因为靶点在提高鸡PGCs体外建系效率中的应用
CN116286612B (zh) * 2023-03-30 2023-10-20 广东省农业科学院动物科学研究所 以mTOR、SMAD信号通路和p53基因为靶点在提高鸡PGCs体外建系效率中的应用

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