Production Of Chimeras Research Articles

Chimera and other fertilization errors

The finding of a mixture of 46,XX and 46,XY cells in an individual has been rarely reported in literature. It usually results in individuals with ambiguous genitalia. Approximately 10% of true human hermaphrodites show this type of karyotype. However, the underlying mechanisms are poorly understood. It may be the result of mosaicism or chimerism. By definition, a chimera is produced by the fusion of two different zygotes in a single embryo, while a mosaic contains genetically different cells issued from a single zygote. Several mechanisms are involved in the production of chimera. Stricto sensu, chimerism occurs from the post-zygotic fusion of two distinct embryos leading to a tetragametic chimera. In addition, there are other entities, which are also referred to as chimera: parthenogenetic chimera and chimera resulting from fertilization of the second polar body. Furthermore, a particular type of chimera called 'androgenetic chimera' recently described in fetuses with placental mesenchymal dysplasia and in rare patients with Beckwith-Wiedemann syndrome is discussed. Strategies to study mechanisms leading to the production of chimera and mosaics are also proposed.

Production of chimeras by aggregation of embryonic stem cells with diploid or tetraploid mouse embryos

The production of mouse chimeras is a common step in the establishment of genetically modified animal strains. Chimeras also provide a powerful experimental tool for following cell behavior during both prenatal and postnatal development. This protocol outlines a simple and economical technique for the production of large numbers of mouse chimeras using traditional diploid morula<-->diploid embryonic stem (ES) cell aggregations. Additional steps are included to describe the procedures necessary to produce specialized tetraploid chimeras using tetraploid morula<-->diploid ES cell aggregations. This increasingly popular form of chimera produces embryos of nearly complete ES cell derivation that can be used to speed transgenic production or ask developmental questions. Using this protocol, mouse chimeras can be generated and transferred to pseudopregnant surrogate mothers in a 5-d period.

A Testis-Mediated Germline Chimera Production Based on Transfer of Chicken Testicular Cells Directly into Heterologous Testes1

In this study, we proposed a testis-mediated germline chimera production system based on the transplantation of testicular cells directly into heterologous testes. The testicular cells of juvenile (4-wk-old) or adult (24-wk-old) Korean Ogol chickens with a recessive pigmentation inhibitory gene, with or without prior culture, were injected (2 x 10(7) cells/head) into the seminiferous tubules of juvenile or adult recipients with White Leghorn with a dominant pigmentation inhibitory gene in a 2 x 2 factorial arrangement. The localization of transplanted cells into the inner space of the seminiferous tubules was confirmed within 24 h after injection. Subsequent testcross analyses showed that 7.8% (5/64) of the recipients had chimeric status in their testes. The periods of time from transfer to hatching of the first progeny with black feathers were 38 and 45 days for adult cells transplanted into an adult recipient, 188 days for adult cells into a juvenile recipient, and 137 days for juvenile cells into a juvenile recipient. Culture of the testicular cells derived both colony-forming and monolayer-forming cells. The colony-forming cells were stained positively for periodic acid Schiff solution, and further reacted with anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies both before and after culture for 15 days. In conclusion, it may be possible to develop the testis-mediated germline chimera production technique, which extends the feasibility of genetic manipulations in avian species.

Detection of High Levels of Recombination Generated During PCR Amplification of RNA Templates

Recombination during the PCR amplification of DNA templates can be a serious problem for those seeking to genotype heterogeneous populations, yet a boon to those seeking to enhance variation during in vitro evolution. Here, the extent to which PCR generates chimeric full-length products was estimated using a powerful restriction fragment-length polymorphism (RFLP) assay involving the use of fluorescently labeled PCR primers. Three different RNA-encoding DNA templates were assayed: (i) one for a group I ribozyme, (ii) one for a 16S ribosomal RNA (rRNA), and (iii) one for a messenger RNA (mRNA). In all cases, the observed frequency of chimeric PCR products exceeded 20%, and longer templates appear to produce more chimeric products. Although two of these templates have the potential to form secondary structures during the PCR, this tendency does not seem to heighten recombination frequency. These results corroborate previous studies that show that the production of chimeras can be best attenuated to a certain extent by varying the extension times in PCR.

Chimeric honeybees (Apis mellifera) produced by transplantation of embryonic cells into pre‐gastrula stage embryos and detection of chimerism by use of microsatellite markers

The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.

Production of medaka chimeras from in vitro cultured embryonic cells

In order to develop an experimental technology employing fish embryonic stem (ES) cells, we attempted to culture cells dissociated from blastula-stage embryos of the medaka (Oryzias latipes). Three types of culture conditions were tested with regard to the culture medium, coating of the culture dishes or addition of conditioned medium. Cells incubated for 1 day under each set of culture conditions were transplanted into recipient embryos to examine whether they retained the ability to form chimeric fish, which was judged according to the body pigmentation. In a control experiment in which intact blastomeres were transplanted, chimeras were produced at a frequency of 32% among survivors. When isolated embryonic cells were dissociated and incubated in a culture medium, most of the cells did not attach to the culture dishes. The frequency of chimera formation by these cells was 15%. Use of gelatin + poly-lysine-coated dishes improved the cell attachment and the cells formed aggregated colonies, but the frequency of chimera formation by these cells decreased to 7%. Further addition of conditioned medium enhanced the cell attachment, and caused the cells to spread individually over the dishes. However, the ability of the cells to form chimeras was lost following this treatment. The results of the present study demonstrate that it is possible to generate viable chimeras from cultured embryonic cells, although further improvement of the culture conditions is necessary to develop a method for culturing fish ES cells.

Rat embryo quality and production efficiency are dependent on gonadotrophin dose in superovulatory treatments

The present study was performed to optimize a superovulation protocol in rats in order to produce a large number of good-quality embryos suitable to develop rat embryonic stem (rES) cells. We first evaluated the ovulation kinetics of three rat strains: Wistar, Fisher and ACI/N. Animals (n=30 per strain) were treated with 50 IU of pregnant mare serum gonadotrophin (PMSG), and ovulation was induced with 50 IU of human chorionic gonadotrophin (hCG) 50 h apart. Next, we evaluated the dose-response curves of PMSG and hCG in Wistar rats in order to obtain the highest number of embryos. The parameters evaluated for superovulation efficiency were: percentage of mated females, percentage of pregnant females and the average number of embryos collected per female. The results of these experiments suggested that the best dose combination was 50 IU for each hormone. Subsequent experiments, again with Wistar rats, were designed to test which of four hormonal combination treatments (30/30, 30/50, 50/30, and 50/50 IU of PMSG/hCG) will produce the largest numbers of good-quality embryos. Embryo quality was evaluated by embryo development uniformity, embryo morphology, embryo survival in an in vitro culture and embryo ability to generate rES-like cells. Results from these experiments showed that 30/50 IU of PMSG/hCG was the treatment that induced the best embryo quality. In conclusion, our results indicated that, in Wistar rats, the most appropriate hormonal combination dose for superovulation protocols with high number of good-quality embryos was 30 IU of PMSG and 50 IU of hCG given 50 h apart. We are performing further studies with rES-like cells produced with the present methodology to evaluate if they are able to participate in the production of germ-line chimeras.

Immunoregulatory Effects of Allogeneic Mixed Chimerism Induced by T-Cell Depleted, Nonmyeloablative Bone Marrow Transplantation on Chronic Inflammatory Arthritis and Autoimmunity Developed in Interleukin-1 Receptor Antagonist-Deficient Mice.

Objective: To investigate the immunoregulatory effects of allogeneic mixed chimerism induced by T-cell depleted, nonmyeloablative bone marrow transplantation (TCD-NMT) on chronic inflammatory arthritis and autoimmunity developed in interleukin-1 receptor antagonist-deficient (IL-1Ra−/ −) mice.Methods: IL-1Ra−/ − mice (H-2kd) were treated with anti-asialoGM1 Ab, TBI 500 cGy, and TCD-NMT from C57BL/6 mice (H-2kb). Engraftment and chimerism were evaluated on peripheral blood (PB), lymph node, and spleen by multi-color flow cytometry. The severity of arthritis was evaluated by clinical score and histopathology. IgG1 and IgG2a subtype of anti-type II collagen (CII) were measured in PB samples. After T cells were stimulated with CII, ovalbumin, and phytohemagglutinin, T-cell proliferation response and cytokines production (INF-g, TNF-a, IL-10, and IL-17) in culture supernatant were assayed.Results: All the transplanted IL-1Ra mice showed marked improvement of arthritis within 3 weeks after transplantation as well as successful induction of mixed chimerism. Mice in mixed chimerism showed higher level of anti-CII IgG1 and lower level of anti-CII IgG2a and weaker T cell proliferative response than in control groups, such as no-treatment and conditioning only without BM rescue. In mixed chimera, INF-g, TNF-a and IL-17 production from CII-stimulated T cells was significantly suppressed and IL-10 production was significantly increased as compared to the control groups.Conclusion: These observations indicate that the introduction of allogeneic mixed chimerism has a strong immunoregulatroy potential to correct established chronic inflammatory arthritis and autoimmunity originating from dysregulated proinflammatory cytokine network.

Cryopreserved Morulae can be used to Efficiently Generate Germline-transmitting Chimeras by Blastocyst Injection

The production of chimeric mice is a complex process, requiring the careful coordination of tissue culture cell growth, production of a large number (30-75) of competent blastocysts and the availability of appropriately timed pseudo pregnant female mice. Failure at any of these steps can impinge upon the rapid production of chimeras. One potential improvement for the efficient generation of chimeric mice would be the utilization of cryopreserved embryos suitable for injection. C57Bl/6 morulae were frozen using a standard 2-step protocol with ethylene glycol as the cryopreservation agent. We determined that cryopreserved morulae could thaw, culture to blastocyst stage in KSOM media and survive injection at rates equivalent to control embryos. Cryopreserved morulae were also equivalent to controls at all later stages in the process of production of chimeric mice, including birth rate, percentage chimerism of resulting animals and ability to produce germline progeny. Hence, cryopreservation of morulae for blastocyst injection is a suitable option to enhance the efficiency of chimeric mouse generation.

“Agouti NOD”: identification of a CBA-derived Idd locus on Chromosome 7 and its use for chimera production with NOD embryonic stem cells

Penetrance of the complex of genes predisposing the nonobese diabetic (NOD) mouse to autoimmune diabetes is affected by the maternal environment. NOD.CBALs-Tyr(+)/Lt is an agouti-pigmented Chromosome 7 congenic stock of NOD/Lt mice produced as a resource for embryo transfer experiments to provide the necessary maternal factors and allow the easy identification of NOD (albino) embryo donor phenotype. CBcNO6/Lt, a recombinant congenic agouti stock already containing approximately 50% NOD genome, was used as the donor source of a wild-type CBA tyrosinase allele. When the incidence of diabetes was assessed after nine generations of backcrossing and one generation of sib-sib mating, significant reduction in diabetes development was observed. No difference in diabetes development was observed in Tyr/Tyr(c) heterozygotes, showing that protection was recessive. Analysis of diabetes progression in another NOD stock congenic for C57BL/6 alleles on Chromosome 7 linked to the glucose phosphate isomerase (Gpi1(b)) locus provided no protection, indicating that the diabetes resistance (Idd) gene was distal to 34 cM (D7Mit346). Approximately 5 cM of the distal congenic region overlaps a region from C57L previously associated with protection when homozygous. The delayed onset and reduced frequency of diabetes in the NOD.CBALs-Tyr(+)/Lt stock is an advantage when females of this stock are used as surrogate mothers in studies involving hysterectomy or embryo transfers. Indeed, a newly developed NOD embryonic stem (ES) cell line injected into NOD.CBALs- Tyr(+)/Lt blastocysts produced approximately 50% live-born mice, of which approximately 11% were chimeric. Presumably because of high genomic instability, no germline transmission was observed.

In vitro culture period but not the passage number influences the capacity of chimera production of inner cell mass and its deriving cells from porcine embryos

Mammalian embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) of the blastocyst. These cells are able to proliferate continuously without differentiation in vitro under suitable conditions. Their capacity of pluripotency in differentiation will be resumed when they are reintroduced into host embryos, when they will contribute to the embryonic development to form chimeric individuals. Manipulation of ES cells has been mainly established from studies in the mouse, and is powerful in the production of transgenic animals. Porcine ICM-derived cell lines possess the same cellular morphology and in vitro behavior as those of murine ES cells, but have lower efficiency in chimera formation when reintroduced into host embryos. This study was to determine the influences of passage number and the duration of in vitro culture on the capacity of porcine ICM-derived cells in the generation of chimeric embryos. The results showed that when passage number of porcine ICM-derived cells was less than 15, there were no detrimental effects on its integration ability. Extending the culture time up to 6 days in each passage of porcine ICM-derived cells impaired its integration capacity into the host blastocyst. Porcine ICM-derived cells cultured for more than 4 days in each passage should not be used for blastocyst injection if high efficiency of chimera production is to be achieved.

The genetic heterozygosity and fitness of tetraploid embryos and embryonic stem cells are crucial parameters influencing survival of mice derived from embryonic stem cells by tetraploid embryo aggregation

The aim of this paper was to determine whether the genetic background of tetraploid embryos contributed to the survival of mice derived from embryonic stem (ES) cells by tetraploid embryo complementation. Twenty-five newborns were produced by aggregation of hybrid ES cells and tetraploid embryos with different genetic backgrounds. These newborns were entirely derived from ES cells judged by microsatellite DNA (A specific sequence of DNA bases or nucleotides that contains mono, di, tri or tetra repeats) and coat colour phenotype and germline transmission. Fifteen survived to adulthood while seven died of respiratory failure. All newborns were derived from outbred or hybrid tetraploid aggregates and no newborns were from the inbreds. Our results demonstrate that the genetic heterozygosity, fitness of tetraploid embryos and fitness of ES cells are crucial parameters influencing survival of mice derived from ES cells by tetraploid embryo aggregation. In addition, this method represents a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.

Novel Reproductive Techniques in Swine Production – A Review

The main objective of modern reproductive technologies in pig reproduction is to increase reproductive efficiency and rates of genetic improvement. They also offer potential for greatly extending the multiplication and transport of genetic materials and the conservation of unique genetic resources in reasonably available forms for possible future use. The development and refinement of these technologies is concentrating on gamete and embryo collection, sorting and preservation, in vitro production of embryos, culturing, manipulation of embryos (splitting, nuclear transfer, production of chimeras, establishment embryo stem cells, and gene transfer) and embryo transfer. Also, the development of these novel technologies is facilitated by modern equipment for ultrasonography, microscopy, cryopreservation, endoscopy, and flow cytometry, microinjectiors, micromanipulators and centrifugation. The real impact on herd productivity will come from combining new reproductive techniques with powerful DNA technologies. The new reproductive techniques will allow a rapid turnover of generations, whereas the DNA technology can provide selection, which does not need phenotypic information when the selection decisions are made.

Dysregulation of granulocyte, erythrocyte, and NK cell lineages in Fli-1 gene–targeted mice

AbstractTargeted disruption of the Friend leukemia integration 1 (Fli-1) proto-oncogene results in severe dysmegakaryopoiesis and embryonic lethality. We used morula-stage aggregation as a strategy to further clarify the hematopoietic defects of the Fli-1 gene-targeted mice. Analyses of lineage expression of Fli-1+/- and Fli-1-/- cells in the peripheral blood and bone marrow of chimeric mice consistently demonstrated reduced numbers of neutrophilic granulocytes and monocytes and increased numbers of natural killer (NK) cells. Transplantation studies using sorted Fli-1 mutant cells produced similar findings. Clonal culture studies of bone marrow cells revealed increased numbers of granulocytic and early erythroid progenitors in the Fli-1+/- cells. The sorted Fli-1-/- bone marrow cells revealed specific down-regulation of CCAAT/enhancer binding protein-α (C/EBPα) and C/EBPϵ, and the receptors for granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF), consistent with their critical roles in granulopoiesis. Collectively, these observations suggest previously unknown physiologic roles for Fli-1 in granulocytic, erythroid, and NK cell proliferation and differentiation. Production of chimeras by morula-stage embryo aggregation is an effective way to unravel cell-autonomous hematopoietic defects in gene-targeted mice.

Production of germline chimeric chickens following the administration of a busulfan emulsion

Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.

Simple and efficient production of mice derived from embryonic stem cells aggregated with tetraploid embryos

Six newly derived hybrid mouse embryonic stem (ES) cell lines and two inbred ES cell lines were tested for their ability to produce completely ES cell-derived mice by aggregation of ES cells with tetraploid embryos. Forty-five ES cell-tetraploid pups were generated from six hybrid ES cell lines and no pups from two inbred ES cell lines. These pups were found to have increased embryonic and placental weights than control mice. Twenty-two pups survived to adulthood and produced normal offsprings, and the other 23 pups died of several reasons including respiratory distress, abdomen ulcer-like symptoms, and foster failure. The 22 adult ES cell-tetraploid mice were completely ES cell-derived as judged by coat color and germline transmission, only two of them was found to have tetraploid component in liver, blood, and lung as analyzed by microsatellite loci. Our data suggested that genetic heterozygosity is a crucial factor for postnatal survival of ES cell-tetraploid mice, and tetraploid embryo aggregation using hybrid ES cells is a simple and efficient procedure for immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps.

181 MOUSE TRIPLETS DEVELOPED FROM SINGLE BLASTOMERES OF AN 8-CELL-STAGE EMBRYO SUPPORTED WITH TETRAPLOID EMBRYOS

Chimera production using altered ES cells became a key tool for generating transgenic mice. However, chimeras are more than just a tool for making mouse mutants; they play a crucial role in analyzing the biological effects of genetic changes. Chimeras can be made by combining two whole 8-cell embryos or by combining subsets of blastomeres of two cleavage stage embryos. Because, at these stages, the early embryonic cells are not yet restricted in their lineage potency, they are equally capable of contributing to the inner cell mass or the trophectoderm. Pluripotency of single blastomeres of the 4-cell and 8-cell mouse embryo has been proved indirectly by aggregating them with carrier blastomers of a different genotype, giving rise to chimeric blastocyst (Tarkowski et al. 1967 J. Embryol. Exp. Morphol. 18, 155-180). In our study we wanted to demonstrate that a single blastomere of an 8-cell stage embryo, supported with tetraploid embryos at the 4-cell stage, is capable of developing into a healthy animal. When tetraploid embryos are used to make chimeras together with diploid cells, tetraploid cells rarely contribute to the embryo itself, but contribute mainly to the primitive endoderm and the trophectoderm, so in this case the newborns will be derived only from the diploid blastomers used (Nagy et al. 1990 Development 110, 815-821). We produced chimeras by aggregating a single blastomere [(2n)(1-cell)] derived by combining sexed, GFP-expressing, diploid 8-cell stage embryo (Hadjantonakis et al. 1998 Nat. Genet. 19, 220-222) with either a sexed diploid 7-cell embryo [(2n)(7-cells)] (one of the 8-cells was taken for sexing) or a non-sexed tetraploid embryo [(4n)(4-cells)]. The aggregates were cultured in vitro and transferred as blastocysts to the uteruses of pseudo-pregnant females. Fetuses were removed by Caesarian section and raised by lactating foster mothers. From the transferred 84 [XY(2n)(1-cell)]/[XX(2n)(7-cells)] aggregates we obtained 12 (14.3%) newborns, 11 (91.7%) males and one (8.3%) female. From the transferred 27 [XY(2n)(1-cell)]/[(4n)(4-cells)] aggregates, where one XY blastomere was combined with a tetraploid embryo, we obtained 7 (25.9%) male newborns: 1 triplet and 2 pairs of twins. We also transferred four [XX(2n)(1-cell)]/[(4n)(4-cells)] chimera embryos, where an XX-blastomere was aggregated with a tetraploid embryo; we obtained a set of living female triplets (75%) from this aggregate. We demonstrated that a single blastomere of the 8-cell-stage embryo is capable of developing into a living newborn. We obtained identical pups from three blastomeres isolated from the same embryo (triplets). This way we produced single blastomere clones and could control the sex of the new generation. This research was supported by grants from OTKA T037582 and RO-1/2002 Intergovernmental S&amp;T cooperation program.

Mouse-chick neural chimeras

Embryonic chimera production was used to study the developmental processes of the mouse nervous system. The difficulty of performing in situ transplantation experiments of neural primordium of mouse embryo was overcome by isotopic and isochronic grafting of mouse neural tube fragments into chick embryo. Mouse neural tube cells differentiated perfectly in ovo and neural crest cells associated with the grafted neural tube were able to migrate and reach the normal arrest sites of host neural crests. Cranial neural crest cells penetrated into chick facial areas and entered into the development of dental bud structures, participating in vibrissa formation. Depending on graft level, in ovo implanted mouse neural crest cells formed different components of the peripheral nervous system. At trunk level, they located in spinal ganglia and orthosympathetic chains and gave rise to Schwann cells lining the nerves. When implanted into the lumbosacral region, they penetrated into the enteric nervous system. At the precise 18-24 somite level, they colonized host adrenal gland. Mouse neural tube was involved in the mechanisms required to maintain myogenesis in host somites. Furthermore in ovo grafts of mouse cells from genetically modified embryos, in which many mutations induce early death, are particularly useful to investigate cellular events involved in the development of the nervous system and to identify molecular events of embryogenesis.

Detection and characterization of primordial germ cells in pheasant ( Phasianus colchicus) embryos

The developmental similarity between the chicken and pheasant ( Phasianus colchicus) allows the novel biotechnologies developed in the chicken to be applied to the production of transgenic pheasants and interspecies germline chimeras. To detect pheasant primordial germ cells (PGCs) efficiently, which is important for inducing germline transmission, the ultrastructure of PGCs and their reactivity to several antibodies (2C9, QB2, anti-SSEA-1, and QCR1) and periodic acid-Schiff’s solution (PAS) were examined. To obtain PGCs, blood was taken from embryos incubated for 62–72 h or from gonads from embryos incubated for 156–216 h. The PGCs collected from both sources had the typical ultrastructure of pluripotent cells: a large nucleus with a distinct nucleolus, a high ratio of nuclear to cytoplasmic volume, and a distinct cytoplasmic membrane. In comparing the morphology of PGCs collected from different sites, more mitochondria and better-developed membrane microvilli were found in gonadal PGCs than in circulating PGCs. The nucleus of gonadal PGCs was flattened and had a large eccentrically positioned nucleolus. Of the antibodies tested, only QCR1 antibody reacted with an epitope in pheasant PGCs, and no specific signal was detected to other antibodies. The temporal change in the PGC populations in the blood and gonads of embryos was examined. In blood, the population was greater ( P < 0.0001) in embryos incubated for 64 h than in embryos incubated for 62 or 66–72 h (31.4 versus 5.6–16.2 μL −1). In embryonic gonads, the number of PGCs increased continuously from 156 to 216 h of incubation (193–2,718 cells/embryo), although the ratio of PGCs to total gonadal cells did not change significantly (0.50–0.61%). In conclusion, pheasant PGCs have typical germ cell morphology and possess the QCR1 epitope. Circulating blood and the gonads of embryos incubated for 64 and 216 h, respectively, are good sources of PGCs.

Production of Interspecific Germline Chimera between Quail and Chicken Utilizing the Cells from Central Disk of Blastoderm and Germinal Crescent Region

Production of interspecific germline chimera between quail and chicken was carried out using the cells from the central disk (CD) of the area pellucida in quail blastoderm (stage X) and the germinal crescent region (GCR) of quail embryo (stage 7-8). The cells of CD and GCR were dispersed and injected into the subgerminal cavity of chicken blastoderm (stage X). Injected eggs were incubated for 7 days or to hatching. To detect quail genomic DNA, the polymerase chain reaction was used. In day-7 embryos, quail DNA was detected in 4 gonads and 6 brains from 55 survived embryos received quail CD cells, and 9 gonads and 8 brains from 68 survived embryos received quail GCR cells. Quail DNA was also detected from eggs or semen of adult female or male chicken hatched from eggs received quail CD or GCR cells. CD and GCR cells as the donors showed the possibility to produce the interspecific germline chimera but the necessary improvement and the immunoreactive problem were suggested.