Method for producing of bird's gametes

FIELD: biotechnology and poultry keeping, in particular, production and collection of bird's gametes.

SUBSTANCE: method involves obtaining embryo stem cells from donor-kind birds and introducing stem cells in-ovo to recipient-kind birds, with recipient-kind birds differing from donor-kind birds; incubating recipient-kind cells to pecking stage; growing to sexual maturity phase and collecting gametes of donor-kind birds from recipient-kind birds.

EFFECT: intensified production of spermatozoa of slowly growing-up birds and reduced expenses for keeping of producing male flock.

11 cl, 1 tbl, 15 ex

The scope of the invention

The present invention relates to methods of transfer of embryonic stem cells for the birds to produce their gametes. These methods are applicable for the protection of endangered species of birds, to reduce the time required to obtain sperm from the slowly maturing birds, such as turkeys, reduce maintenance costs stud manufacturers and changes in sex ratio in herds descendants (i.e. increasing the efficiency of production).

Background of the invention

To more easily obtain gametes certain species of birds would be extremely useful for veterinary birds and poultry farms. For endangered species, such as American pie, will be extremely useful to have a ready stock of men's sperm. For commercial poultry, such as Turkey, it would be desirable faster and more economical the obtaining of male sperm. For meat production, it is desirable to have methods of altering the sex ratio of birds in the flock. Thus, there is a need for new methods of obtaining sperm birds.

Chimeras are called organisms are composed of cells received more than one zygote. Experimental chimeras were used to study the interaction between cells and analysis of the descendants of the cells in the process of the development (.McLaren, Mammalian Chimeras. Cambridge University Press, Cambridge (1976)). When chimeras receive, using material derived from very early embryos, developing organisms, containing a complete mixture of somatic tissues. If the starting material includes cells of the early embryo or their predecessors, the resulting individuals will produce gametes with genotypes of both donor and recipient. In addition, the chimeras can be intraspecific, i.e. between zygotes of the same species, or between species, i.e. between two different types.

Embryonic stem cells (ESCs) birds, as the embryonic cells of other vertebrates, are vaginalnyj origin and must endure a difficult journey to reach the gonads. Transfer blastodermal cells and embryonic stem cells formed embryonic chimeras birds.

Reynaud (J. Embryol. Exp. Morphol. 21:485-507 (1969)), a pioneer in obtaining embryonic chimeras birds reported obtaining embryonic chimeras Turkey-chicken by intravascular migration split sickle cell body of the embryo in the pre-sterilized chicken embryo (through the impact on the Crescent-shaped body of the recipient UV). ESK obtained by mechanical separation endoderm sickle body of the embryo (stage 5)were injectively in the blood vessels of the embryo Caplan is a (3-5 days of incubation). Before injection, the recipient embryos were sterilized at stages 8-10 (H&M) ultraviolet radiation; however, sterilization was not completed. ESK turkeys in the chicken embryo was determined solely on the basis of their relative nucleoplasm. This method of identification was complicated and delicate, and could not be used to actively dividing ESK turkeys, so as dividing embryonic cells gave aberrant nucleoplasmic ratio. In subsequent studies moved ESK allowed to ripen in the gonads of the host, and obviously, they could give rise to gametes, but they were not applicable for fertilization (Wilhelm Roux Arch. Dev. Bio. 179:85-110(1976)). These sperm were unable to fertilize Turkey eggs. They were fertilized chicken eggs, but normal development occurred. Chicken spermatozoa were able to activate eggs obtained from females of interspecific chimeras, but they did not cause the development of the embryo. When the eggs were fertilized by the sperm of Turkey, they have evolved in abnormal embryos that did not live longer stage 38 (H&M). Reynaud used morphology as the only distinctive characteristic in attempting to distinguish germ cells Turkey from embryonic chicken cells. Only one morphology is insufficient to determine the chimeras and must be supplemented with other markers. The AOC is E. this according to Aig-Gill and Simkiss (Brit. Poul. Sci. 32:427-438 (1991)), the presence of gametes Turkey has not been shown in test matings. Thus, there is a need for new methods of production and transfer of gametes birds.

The invention

The method of production and semen collection birds involves the following stages: obtaining embryonic stem cells from birds species of the donor; the introduction of embryonic stem cells to birds species-recipient in ovo; incubating birds species-recipient before hatching; and then collecting the sperm of view of the donor on the type of recipient. For example, as a donor may be American crane, and the recipient can be a Sandhill crane. In another example, a donor may be Turkey, and the views of the recipient may be a chicken.

In birds, unlike mammals, the male is homogametic sex (ZZ) and the female is the heterogametic sex (Zw). Therefore, in birds, the female determines the sex of your offspring, because it produces eggs bearing Z or w chromosome. Thus, as shown below, when transferring the embryonic stem cells of the male embryo master of the females, the percentage produced by this host eggs bearing the Z-chromosome, increases as increases and the percentage of descendants of males. The increase in the percentage of male descendants in flocks of broilers is economically desirable due to a corresponding increase in sootnoshenie the food conversion and greater efficiency of production of meat, achieved in this way.

Thus, the second aspect of the present invention is a method of increasing the number of birds (males), derived from the egg mass, which includes stages: introduction of the female bird in ovo embryonic stem cells male (ZZ); incubation the female bird to hatch; the cultivation of female birds to sexual maturity; and then the multiplication to obtain the mass of fertile bird eggs (with a ratio of males/females in eggs obtained from this bird, larger than in the case when the embryonic stem cells of the male did not enter the female bird in ovo). In the typical case, then this method involves the step of incubating the mass of the bird's eggs before hatching (with a ratio of males/females obtained in the mass of eggs, large, than when the embryonic stem cells of the male did not enter the female bird in ovo). Female birds may be of any suitable type, such as chicken or Turkey, and injected embryonic stem cells, preferably from the same species as the female, which they are administered.

The above and other objects and aspects of the present invention are explained in detail in the description below.

Detailed description of preferred embodiments of the present invention

The terms "bird" or "bird species" is used here to refer to any kind of birds, including chicken, Turkey, duck,geese, quail, pheasant, ostrich, but are not limited to. Any of the many other species of birds can be used to implement the present invention, in particular when it is used for protection of endangered species, such as American pie (where the views of the recipient would be a Sandhill crane).

The term "egg" is used in relation to avian eggs containing live embryos of birds.

The term "embryonic stem cells" or "ESC"is used in relation to the most differentiated diploid cell line of the embryo, which eventually develop into haploid gametes (sperm and egg).

The term "antibody SSEA-1" refers to the antibody, preferably a monoclonal antibody, which is specific connected with stagespecific embryonic antigen-1 (SSEA-1; (M.Buehr Exp. Cell. Res. 232:194-207 (1997)). SSEA-1 is a carbohydrate epitope, deterministic communication galactose β1→4 fructose α1→3N acetylglucosamine (.Gooi et al., Nature 292, 156-158 (1981)). Monoclonal antibody for SSEA-1 was developed by fusion of murine myeloma cells with spleen cells of a mouse that was immunized cells teratocarcinoma F9 (D.Solter and B.Knowles, Proc. Natl. Acad. Scl. USA, 75, 5565-5569 (1978)). Antibody SSEA-1 is known as an immunohistochemical marker of germ cells of birds (L.Karagenc et al., Dev. Genet. 19, 290-301 (1996)). In private the tee, the preferred clone 480 MS, which can be obtained from Developmental Studies Hybridoma Bank, University of Iowa, Iowa city, Iowa, USA.

Embryonic stem cells can be obtained and receptivity for carrying out the present invention by any suitable spacebike, store, freeze, kulturarbeit and the like before use, if necessary. For example, embryonic stem cells can be extracted from embryonal donor on the corresponding embryonic stage (see, for example (V.Hamburger and H.L.Hamilton, A series of Normal Stages in. the Development of the Chick, Journal of Morphology, 68, 49-42 (1951) (These stages relate to H&N stages) stage 4 or embryonic stage sickle of the body to the stage 30, with cells taken from blood or gonads at later stages). Embryonic stem cells, in most cases, twice the size of somatic cells and they are easy to distinguish and to separate based on size. Male (or homogenate) embryonic stem cells (ZZ) can be distinguished from the heterogametic embryonic stem cells (Zw) by any suitable means, such as collection of embryonic cells from a specific donor and typing, other cells from this donor, the collected cells of the same chromosome type, as typed cells. Cells may be prepared for administration to animals through cell division (i.e. fur the technical division) and the subtle blending of cells to a pharmaceutically acceptable carrier (e.g., phosphate buffer saline). Embryonic stem cells-mainly embryonic stem cells of the gonads or embryonic stem cells of the blood ("gonads" or "blood" with respect to their tissue or origin in the original embryonic donor), most preferably embryonic stem cells of the gonads. Input embryonic stem cells can be heterogametic (Zw) or homogametic (ZZ) depending on the specific object introduction. ESK preferably in a physiologically acceptable medium, preferably at pH from about 6 to 8 or 8.5; in a suitable amount to achieve the desired effect (for example from 100 to 1000 ESC on the embryo). ESCs can be entered without other ingredients or cells, or other cells or the ingredients may be introduced together with the ESC.

Introduction embryonic stem cells of the animal to the recipient in ovo may be held at any appropriate time, when the ESC can still migrate to the developing gonads. In General, it is preferable that the introduction was carried out from the stage 13 or 14 to stage 18 (H&M) embryo development, and most preferably at stage 15. For the chicken, so the time of the introduction are the days 1, 2, 3 or 4 of embryonic development, most preferably from 2 to 2.5. Usually the introduction is carried out by injection into the AC the second target region, such as the region defined by the amnion (including the embryo), yolk SAC, etc. Injection directly into the embryo (including the body wall of the embryo) are preferred intravascular or nutricionista injection into the embryo is particularly preferred. The methods of the present invention can be effected with or without prior sterilization birds recipient in ovo. ("sterilization" means bringing to the condition in which the production of gametes is almost impossible). In the preferred embodiment of the present invention embryonic stem cells administered to the recipient in ovo, without prior sterilization. When donor gametes collected from such recipient, they can collect in a mixture with donor gametes and used as a mixture or mixture that has been treated to increase the proportion of donor gametes.

The introduction of ESC can be produced through the introduction of ESC as such or through the introduction of cells predecessors, which develop in ESK already in the subject (in particular, when the invention is applied to change the sex ratio in the offspring). For example, the introduction may be made by injection bird blastodermal cells when blastodermal cells differentiate into embryonic stem cells in the bird in vivo.

When embryonic stem is the first cell is used to produce and collect gametes birds (sperm, egg cells), embryonic stem cells injected in ovo appearance-the recipient is different than the donor, from which were obtained ESC. The recipient then incubated to hatch and grow to sexual maturity and collect the sperm or the egg type of donor, all in accordance with standard spacebecame. For example, in the case of endangered species, species-the donor may be American crane and views of the recipient can be a Sandhill crane. In another example relating to commercial poultry production, as the donor may be a Turkey, and the views of the recipient may be a chicken.

When used to increase the number or proportion of birds (males), displayed from a group of eggs, the invention includes the introduction of the female bird in ovo embryonic stem cells of the male. The sex of the bird in ovo may be pre-defined or determined after hatching. The bird then incubated until hatching, when necessary, determine the sex of the bird, grown to sexual maturity and bred, crossing with a suitable male by the manufacturer in accordance with known spacebecame. The whole mass of fertile eggs, pending this bird, then harvested and the usual way incubated until hatching, and the resulting birds are grown for at least two or three weeks. The ratio of males to females eggs (or birds) proizvedeniya females more than those obtained in the absence of the introduction of embryonic stem cells of the male the female in ovo. Such methods are usually used for bird species that are grown for meat production, such as chickens, turkeys, ducks, etc.

Introduction embryonic stem cells in ovo can be carried out by any suitable technique both manually and automatically. The preferred injection. The mechanism in ovo is not critical, but preferably, the method is not excessively damaged tissues and organs of the embryo or extraembryonal shell surrounding it, so that this processing is not reduced excessively, the number of hatched Chicks. Syringe for subcutaneous injection, suitable for needles around from room 18 to room 26 convenient for this purpose. Depending on the exact stage of development and position of the embryo, the needle is long in one inch can get as a liquid over a chick and the chick. Pilot hole may be punched or drilled in the shell before the introduction of the needle, to prevent damage or dulling of the needle. If necessary, the egg can be sealed by a material substantially impervious to bacteria, such as wax or the like, to prevent the subsequent entry of undesirable bacteria. It seems that the high-speed injection system DL the embryos of birds is particularly suitable for use the present invention. Numerous devices of this kind are available, a model can serve as a system EMBREX INVOJECT™ (described in U.S. patent No. 4681063 and 4903625 Hebrank) and U.S. patent No. 4040388, 4469047 and 4593646 Miller. Open all of the U.S. patents cited here, included here in its entirety by reference. All such devices being adapted for practice of the present invention, include injector containing embryonic stem cells, rezeptfreie, as here discussed, with the injector located to inject into the egg, the roaming device with the relevant provision of the eggs, as discussed above. In addition, injicerade the device may be equipped with a sealing device to seal the holes in the egg after the injection.

The present invention is described in more detail in the following non-exhaustive Examples.

Examples 1-8

Material and methods :

EXAMPLE 1

Selection and verification of plasmids.

Transformed DH5 alpha cells derived other Mbacke, were deposited at the laboratory of the Cup containing the antibiotics ampicillin (20 µg/l) + methicillin (80 µg/l) and left to grow overnight at 37°C. Six individual colonies were selected and grown overnight in 10 ml of laboratory cups containing the above antibiotics. Plasmid DNA isolated is from 6 different colonies, using preparatory mini Protocol (Qiagen. In order to verify the identity of this plasmid, undigested plasmid, linearized plasmid (EcoR 1) and the double cleavage (EcoR I + Hind III)were separated on 2% agarose gel. Two of the six colonies containing the insert were then used for large-scale isolation of plasmid (Qiagen). Undigested parent plasmid (RIS), undigested recombinant plasmids, linearized plamid (EcoR I/ Hind III/ BamH I) and double-digested plasmid (EcoR I + Hind III and EcoR I + BamH I) were separated on a 2% gel to confirm the identity of the isolated plasmids.

EXAMPLE 2.

The tagging sample TM1 using PCR.

Was synthesized primer pair on the basis of their ability to amplify the insert in multiglandulosa site's parent plasmids figure 18. They were M13 puc - reverse = 5' AAC sintering shop TAT Yai UAT and M13 puc - direct - 5' GTA AAA the archives CGG CCA GT. Optimized PCR mixture consisted of 3 mm MgCl₂in Taq buffer (Idaho Tech); 0.5 µm of each primer, 50 ng DNA (TM1) ring denatured plasmid, 5 units of Taq polymerase (Promega), 10 μl of PCR aiming (dig-labeling mix (Boehringer, Mannheim). The volume of the reaction mixture is brought to 100 μl with sterile water, preparation of PCR consists of an initial denaturation at 96°C for 5 min followed by 30 cycles consisting and the denaturation (94° (C) within 45 seconds recovery (50° (C) for 55 seconds, and subsequent elongation at 72°C for 60 seconds. PCR was performed in a Mini Cycler model PTC 150 (MJ Research Inc., Massachusetts). After amplification, the entire sample was subjected to electrophoresis in a 2% gel. Labeled box was suirable from the gel using a Qia set quick gel extraction (Qiagen) according to the manufacturer's recommendations. The sample was kept at -20°and used for in situ DNA dot blot hybridization. Before storing estimated the amount of DIG-labeled DNA in accordance with the system management Genius for hybridization to filter (Boehringer, Mannheim).

EXAMPLE 3

Dot-blot hybridization

To test the accuracy, sensitivity and specificity-insert TM1 serial dilution of the DNA of the male and female turkeys (0-50 ng), the DNA of the male and female chicken (0-2 μg) and the parent plasmid containing the insert TM1 (10 ng - 1 PG) was denaturiruet and were applied to nitrocellulose paper. The spot was heated at 80°within hours and then used for hybridization. Pre-hybridization and hybridization was performed using the procedure of Engler-Blum [Anal Biochem. 210:235-244 (1993)). Hybridization was performed overnight at 68°C; used a sample with a concentration of 2.2 ng cdnc in the sample in ml.

After hybridization and thorough washing, the blot was placed in a wash in the morning is (0.1 M maleic acid; 0.15 M NaCl pH 7.5). The membrane was incubated in blocking solution (wash buffer + 3% Tween 20) for 30 minutes and then placed in a blocking solution containing the conjugate antidigoxigenin alkalinity for half an hour. The membrane is then washed twice in wash buffer and then incubated in the indicator solution (0.1 M Tris HCl; 0.1 M NaCl; 50 mm MgCl₂; pH of 9.5). In the final hybrids were recorded using chemoluminescent substrate COP-STAR™ (Boehringer-Mannheim, Germany). Spots were subjected to x-ray film for at least 5 minutes.

EXAMPLE 4

The interspecific Turkey-chicken embryonic germline chimeras.

Fertilized eggs were incubated at a 38.5°for 8-8,5 days (stage 27-28 H&M). Embryos analyzed to obtain the gonads. Gonads were collected in DMEM and 10% FBS and were dispersively pass through a needle of No. 30. The cells were konturirovany in DMEM 10% FBS prior to connection (3-5 [d] days?). Stromal cells were dispersibility and formed a continuous layer, while embryonic stem cells loosely attached to the stromal cells. Embryonic stem cells was carefully collected with a pipette and counted. Approximately 150-300 cells in 2-5 ál of medium was injected into the sinus terminalis 60 or 72-hour chicken embryos. Then the embryos were incubated in 100 mm Petri dishes or in their own shell with a 38.5°during the course the e 2-5 days. After incubation, DNA was extracted from embryos (n=18) and used for dot-blot analysis with dig-labeled sample TM1.

Example 5

In situ hybridization.

The in situ hybridization posting on paraffin sections and krisrose. This procedure is based on the Protocol Rolighed and Lindbergh (see J. Rolighead, Direction of II HPV DNA in paraffin embedded laryngeal tissue with DIG-labeled DNA probe. Ir. Non-radioactive In Situ Hybridization Application Manual Boenringer Mannheim Second Edition, pp 122-125 (1996)) with some modifications.

Manufacture of paraffin sections: Gonad extracts from embryos turkeys (9th day) and chicken embryos at appropriate stages, fixed overnight in 4% formaldehyde at 4°C. Gonads washed in PBS three times for a total of 90 minutes. Then dehydrate them, pour in the wax and make the sections (10 μm). The slices are placed on a glass Probe-On Plus^tm(Fisher Scientific). The slices are heated at 60°C for 30 minutes, remove the paraffin in xylene and rehydration in a gradient of ethanol 99% water. Slices treated with proteinase K (50 μg/ml and 100 μg/ml) in TES (50 mm Tris HCl pH 7.4; 10 mm EDTA and 10 mm NaCl) for 12 to 25 minutes at 37°and at room temperature.

Making crisisof: Area of the trunk of 8.5 day embryos turkeys fixed overnight at 4°With a 4% solution of formaldehyde in PBS. Were tested with different concentrations of proteinase K in TES from 0 to 45 µg/ml for 10, 15 and 20 minutes at 37°C. Optimal the conditions for proteolytic processing of embryonic tissues were: 1,25 µg/ml at 37° C for 15 minutes.

Preparation of cocktail samples and blind [control] cocktail: Cocktail samples consisted of 10 μl of 50 × solution of Danaharta, 50 μl of dextran sulfate (50%), 10 ál of DNA salmon sperm (9.4 mg/ml), 100 ál 2C XSSC, 500 ng TM1 sample labeled with digoxigenin, and brought with distilled water to a final volume of 250 μl. At the end of the cocktail was added 250 μl of formamide. Blind cocktail contained all the above components except the labeled TM1 samples. The cocktail was mixed by turbulence [vortexing] and stored at -20°C.

Hybridization: After proteolytic cleavage as paraffin and crisisi were fixed in 0.4% paraformaldehyde for 5 minutes at 4°C. Then the sections were washed in distilled water (5 minutes) and dried in air. Then 10 or 15 ál of cocktail samples or blind cocktail (negative control) were added to each slice. Before denaturation at 95°C for 6 minutes, the sections were covered with covered with silicone cover glass. The slides were then placed on a minute on ice and placed in a moist chamber for 16-20 hours at 42°C. money Carefully and check hybrids were similar to those described in Rolighed and Lindbergh (see above), except ready-to-substrate of alkaline phosphatase NBT/BCIP (Amresco), which is used for registration of hybrids. After the registracii drugs dust colored aqueous eosin for a few seconds, and then rinsed. The samples were mounted in aqueous medium prepared from 10 g of gelatin dissolved in 60 ml of water at 70-80°to which was added 70 ml of glycerol and 1 ml of phenol.

Example 6.

The interspecific embryonic chimeras Chicken - Turkey.

The embryos of chickens breed Barred Rock incubated until stage 23-25 (H&M). Genital ridges ten embryos together with some adjoining tissues were placed in DMEM with addition of 10% FBS, glutamine, antibiotic and antifungal solution. They then twice washed in PBS and incubated in 0.02 EDTA at 37°within fifteen minutes. Added a fresh environment and combs shared with needles. The entire cell suspension was collected in a 15 ml test tube and allow the clumps to settle for a couple of minutes. Cell suspension was collected and centrifuged at 1500 rpm for 5 minutes the Medium was removed and cell viability was determined using [trypan blue] exception. Selected aliquots of cell suspension and stained using antibodies SSEA-1, to determine the number of embryonic cells. Approximately 5 μl of cell suspension containing 25-30 ESK (the percentage of PGCs in the cell suspension was approximately 3.2 percent; injected into a blood vessel of each embryo turkeys Nicholas (n=10) development stages 13-14 (H&M). Embryos were incubated in glass cups, closed plastic is Lenka when 37,5° With up stages 21-25. The entire body of the embryo recipients were fixed in 4% paraformaldehyde overnight, washed three times in RBS just over 90 minutes, were placed in gelatin/sucrose froze and did the cuts. As ESK gonads turkeys SSEA-1 negative and ESK gonads chicken SSEA-1 positive, antibody against SSEA-1 can be used to determine the transfer of donor PGCs in embryonic chicken germline chimeras.

EXAMPLE 7. The interspecific Turkey-chicken embryonic germline chimeras.

Fertilized eggs were incubated at a 38.5°C for 8 8.5 days (stage 27-28 N&H). Embryos analyzed to obtain the gonads. They were placed in PBS and incubated in 0.02% EDTA at 37°C for 20 minutes. Added a fresh environment and crests gently shared with needles. Collected the entire cell suspension and centrifuged at 1500 rpm for 5 minutes. The medium was removed and determined cell viability. The entire cell suspension were prepared at 37°in DMEM + 10% FBS for 6-7 hours. After kulturarbeit unattached cells were gently collected and centrifuged. Then 2-3 ál of cell suspension, containing approximately 150 ESK, was injected into the blood vessels of the chicken embryos at stage 14 (H&M). Then eggs-recipients were sealed and incubated at 37,5°C. Embryos recipients were collected at different stages in which ubali from stage 19 and stage 25. Embryos were washed in PBS twice; total time varied depending on the thickness of the embryo. Embryos were immersed in 50% ethanol and placed in paraffin. With sections removed paraffin, rehydratable and washed in PBS.

Control for spacebike double staining (see below) was cross sections of the two chicken embryos stage 26 and two embryos turkeys stage 24. Forty-two slice genital chicken and all serial sections genital turkeys were painted.

Only 8 chick embryos recipient were made of a series of slices. Five of the eight embryos were fixed at stage 19 and 20. Two embryos were fixed at stage 22 and 23. Last embryo was fixed at stage 25. Most slicers stages 19 and 20 were used for double staining. Only every second slice embryo stages 22, 23 and 25 were used for double staining.

Example 8.

Double staining with antibody SSEA-1 and dye PAS. Immunohistochemical studies were performed using a set of ABC-AR (Vector Laboratories, Burlingame, California). The slices are washed three times in PBS, all within 30 minutes. Then they were blocked in 1.5% goat serum in PBS for 20 minutes to kill nonspecific binding. Then the slices were incubated for one hour with the primary monoclonal antibody for SSEA-1 (clone MS 480, obtained in Developmental Stuies Hybridoma Bank, University of Iowa, Iowa city, Iowa). After washing in PBS the sections of embryos were incubated with biotinylated secondary antibody (30 minutes), then washed in PBS, and incubated in the reagent Vectastain ABC-AP (30 minutes). After the final washing in PBS, they were stained in the substrate of alkaline phosphatase NBT/BCIP (Amresco, Solon, Ohio) for 20 minutes.

Following the process immunohistochemical staining, sections were washed with tap water and placed in periodno acid for 6 minutes. Then the sections were washed in water for 10 minutes and stained with the reagent Chiff within 15 minutes. After washing in tap water, the sections were mounted in aqueous medium.

EXAMPLES 9-15

Results.

Matzke and others (Chromosoma 102: 9-14 (1992)) describe a repeating sequence of DNA that is enriched on the microchromosomes turkeys. It is formed by repetitive element 41 nucleotide pairs, represented 5% of the genome (approximately 2.2×10⁶copies in a diploid genome of the cell). Thus, this species-specific DNA sequence used in DNA-DNA hybridization, to check whether you can use it to identify DNA turkeys in the embryos of chickens.

EXAMPLE 9.

Selection and verification of plasmids.

Analysis of gel electrophoresis showed that the parental plasmid puc 18 was 2,69 TPN, while the linearized re obinata plasmid, received from other Matzke was about 2.8 TPN in length. Double cleavage of plasmid DNA from the colonies No. 2 and 5 have identified the insertion of approximately 0.15-0,17 TPN. This confirmed that we received transformed DH5 cells contained the appropriate rekombinantnog plasmid. The plasmid contains a fragment of TM1 (149 mo), consisting of three copies of a specific turkeys repeat 41 mo.

EXAMPLE 10

PCR labeling sample TM1.

PCR amplification of circular plasmid DNA with nucleotide labeled with digoxigenin, led to amplificatoare product about for 0,19 0,20 TPN. The increase in the size of the amplification product in comparison with insert (0,15-0,17 TPN) has occurred, probably due to the inclusion of multiple DIG-labeled nucleotides. PCR-labeled sample was then used for experiments on the dot-blot hybridization and DNA-DCC In situ hybridization.

EXAMPLE 11

Dot-blot hybridization.

The results of dot-blot hybridization (data not shown) showed the same intensity of communication samples with DNA samples turkeys males and females. This is confirmed by the fact that this test is not specific to gender. Hybridization was done on serial dilutions of DNA turkeys from 500 ng 0 ng DNA turkeys. In the sample registered a total of 0.30 ng DNA turkeys. Hybridization with 0-2 µg DNA chicken, ka is male, and females, confirmed that the sample was species-specific and did not correlate with the DNA of the chicken. Different concentrations of DNA turkeys (10 ng 0 ng) was mixed with 0-2 μg DNA of the chicken. Hybridization in this mixture of DNA showed that only 1.25 ng DNA turkeys could be marked in 1 µg DNA of the chicken.

Example 12

The interspecific Turkey-chicken embryonic germline chimeras.

Interspecific embryonic germline chimeras Turkey-chicken were not found using the above Protocol, the dot-blot hybridization. The reason for this could be the existence of intrinsic biological barrier that would prevent migration of the PGCs of the gonads turkeys in the gonads of chicken. It could also be due to technical problems, i.e. the procedure (the dot-blot hybridization) was not sensitive enough to register multiple donor embryonic cells in the gonads of chicken. The last reason seems to be more plausible; hence an attempt was made to create a more sensitive sposoby i.e. sposoby in situ hybridization to localize ESK donor in the recipient.

EXAMPLE 13

Analysis of in situ hybridization on sections turkeys.

Theoretically marker system in situ would be a suitable marker to identify donor cells (Turkey) in the Chimera. Because the marker is inside the nuclei, it is universal, it doesn't get in the other the cells and does not affect the development of the embryo recipient. In the present study, the sequence TM1 selectively contacted with the DNA in the nuclei of cells turkeys (data not shown). No positive signal was not observed in chicken cells (data not shown) or slices, incubated with a blind cocktail (data not shown), which indicates that the sample was species-specific, without nonspecific signals. Ideally, the positive control sections of embryos turkeys all cores would be positive. However, only a small percentage of cells stained positive (data not shown). In addition, there was a difference in signal intensity between different populations of cells in the same slice, under the same conditions of enzyme treatment. This shows that this spacebike may give false negative results. Reduce the percentage of false negative results can be achieved by loosening the strictness conditions. However, it may also lead to an increase in false positive results. In embryonic embryonic chimeras cells of the donor would be a very small percentage of the total embryonic slice or cells. Moreover, this marker system defines only a minority of positive cells turkeys. Thus, in situ hybridization with TM1 breakdown will not be an effective way to identify chimeras.

EXAMPLE 14

About what the definition of interspecific embryonic chimeras chicken-Turkey using coloring SSEA-1.

In order to ensure that no technical problems associated with sposobami, interspecific chicken-Turkey embryonic chimeras were obtained by intravascular migration of embryonic cells of the gonads of chicken. Because there are species differences in the expression of SSEA-1 antigen on the ESK gonads chicken and Turkey, suggested that the antibody SSEA-1 can be used to identify embryonic germline chimeras chicken-Turkey. Of the five survivors of four embryos were cut into crisisi. In one of the 4 embryos to the dorsal mesentery embryo turkeys were found 19 SSEA-1 positive cells (data not shown), another 4 chicken embryonic cells labeled SSEA-1, were identified in the genital ridge of Turkey (data not shown). In the second embryo two SSEA-1 positive cells were recorded in the vicinity of the gonads. In the two remaining embryos donor PGCs were not marked.

On the basis of these results it can be concluded that ESC gonads of chicken embryos, starting with the 5 day (the stage in which ESCs are SSSA-1 positive), when the intravascular introduction of embryo turkeys on stage 13, capable of migration, colonization of the gonads and give rise to germline chimeras. Thus, it appeared that the chemoattractant produced by the gonads turkeys, is not species-specific. Also again confirm the Deno, what ESC gonads chicken retain their ability to migrate even after they colonized the gonads. Low efficiency of germline chimeras in this study can be attributed to the smaller number ESK donor-entered the cell suspension.

Example 15

Identification of interspecific embryonic chimeras Turkey-chicken with using colouring SSEA-1 and PAS.

Previous research has shown differences in the expression of SSEA-1 in ESK Turkey and chicken. These antigenic variations associated with standard PAS test can potentially be used to identify germline chimeras Turkey-hen. Research double-dyed slices of chicken embryo confirmed that ESK chicken as positive to PAS and SSEA-1 (data not shown). In control slices of chicken embryo was not found PAS positive and SSEA-1 negative embryonic cells. Double staining 24 stage turkeys through PAS and SSEA-1 confirmed that the ESC turkeys, migratory through the dorsal mesentery and colonizing the gonads, PAS positive and does not Express SSEA-1 epitope (data not shown). Thus, the double staining of embryos chicken and Turkey found that spacebike double staining can be used as a marker to identify embryonic cells turkeys in the gonads of chicken. When using and what Titel SSEA-1 together with the standard dye PAS embryonic chimeras were detected in four of the eight chick embryos-recipients (Table 1). Approximately 24 hours after injection ESK turkeys in the blood vessels of the embryo chicken SSEA-1 negative and PAS-positive embryonic stem cells turkeys were found in chicken embryos. ESK turkeys were recorded together with the Eska chicken in bold coelomic epithelium (data not shown). This epithelium was between coelomic angle and mesonephros, in the field of the future gonads. In older embryos (stage 22 and 23) ESK donor-turkeys were noted in both the embryo-recipient of the chicken. Some germ cells were located in the dorsal mesentery (data not shown), while others migrated further and colonized the chicken gonads (data not shown). Analysis of potential chimeras using spacebike double staining confirmed that the ESC gonads turkeys can be used to produce interspecific chimeras.

Although DNA-DNA hybridization and would is and speciesspecific, this spacebike were unable to identify chimeras. The way the dot-blot hybridization was not sensitive enough to detect the ESC donor, whereas the method in situ hybridization has a high percentage of false negative results associated with sposobami. The way double staining is a successful method for the identification of chimeras Turkey-hen. Based on the results presented above, ESK gonads of chicken or Turkey with intravascular introduction are able to migrate into the gonads and give rise to the germline Chimera. Thus, it appeared that the chemoattractant produced by the gonads of birds, is not species-specific. This also proves that the ESC gonads retain their ability to migrate even after they colonized the gonads.

Getting chimeras Turkey-chicken has wide application. Transfer ESK male turkeys suitable for spermatogenesis turkeys in the gonads of chicken. This can improve spermatogenesis, since the time required to obtain sperm from laying hens 18 weeks, compared to 30-32 weeks in turkeys. The ability to kulturarbeit ESK and get germline chimeras can reduce the number of high-quality manufacturers that are currently required to produce offspring. The possibility of obtaining Turkey sperm from smaller and cheaper birds also Vyg the bottom for poultry.

Experimental chimeras can also give the model studies the interaction between germ and somatic cells of different genotypes on the basis of which it becomes possible to know whether this neighboring cells any characteristics of embryonic cells. This spacebike can also be used for migrating PGCs from species with low fecundity to a more prolific birds and to maintain ESCs in case of an unexpected death or illness or in the case of species of birds, endangered, under natural mating conditions (A.Tajima et al. Theriogenology 40:509-519 (1993)).

The foregoing is illustrative of the present invention and should not be construed as limiting. The invention is defined by the following paragraphs and equivalent provisions included in them.

1. Method for the production and collection of gametes birds, providing stage: obtain embryonic stem cells from birds species donor; such embryonic stem cells referring to the recipient bird in ovo, where the specified recipient is different from the type of donor; incubation specified recipient before hatching; the cultivation of the specified recipient to sexual maturity; and subsequent collection of gametes of the specified kind donor has specified a recipient.

2. The method according to claim 1, where the specified form donor who is American pie.

3. The method of claim 2, where the specified-recipient is a Sandhill crane.

4. The method according to claim 1, where the specified form donor is Turkey.

5. The method according to claim 4, where the specified form of PR is a chicken.

6. The method according to claim 1, where the specified stage injection is performed by injection.

7. The method according to claim 1, where these embryonic stem cells are selected from the group consisting of embryonic stem cells and embryonic gonads blood stem cells.

8. The method according to claim 1, where the specified stage of injection is performed at stages 13 and 18 of embryonic development.

9. The method according to claim 1, where these embryonic stem cells are a pair of chromosomes (Z)determining the gender of male.

10. The method according to claim 1, where these embryonic stem cells have chromosome (w), which determines the gender of a female.

11. The method according to claim 1, where the specified stage injection is performed without prior sterilization specified birds in ovo.