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Overt female sheep-goat chimera produced from the injection of a caprine ICM (Alpine • Nubian) into an ovine blastocyst (Rambouillet X Rambouillet). 

Overt female sheep-goat chimera produced from the injection of a caprine ICM (Alpine • Nubian) into an ovine blastocyst (Rambouillet X Rambouillet). 

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Embryos were surgically flushed from goats and sheep on d 6 and 7, respectively, following the first day of estrus (d 0). After enzymatic removal of the zonae pellucidae, inner cell masses were isolated from caprine blastocysts by immunosurgery. The intact inner cell masses were injected into ovine blastocysts with the aid of a micromanipulator. Tw...

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... The foster mother's species will be selected to be taxonomically closely related, but less endangered. This is a highly experimental approach, which has so far been successfully applied between sheep and goat [138,139], and between sheep embryos [140], but may represent a viable option in the future e especially if combined with artificial gametes derived from induced pluripotent stem cells [46]. ICM exchange has not been attempted so far as a solution to the pressing problem of lacking reproductively healthy recipients in critically endangered species. ...
Article
The ongoing mass extinction of animal species at an unprecedented rate is largely caused by human activities. Progressive habitat destruction and fragmentation is resulting in accelerated loss of biodiversity on a global scale. Over decades, captive breeding programs of non-domestic species were characterized by efforts to optimize species-specific husbandry, to increase studbook-based animal exchange, and to improve enclosure designs. To counter the ongoing dramatic loss of biodiversity, new approaches are warranted. Recently, new ideas, particularly the application of assisted reproduction technologies (ART), have been incorporated into classical zoo breeding programs. These technologies include semen and oocyte collection, artificial insemination, and in-vitro embryo generation. More futuristic ideas of advanced ART (aART) implement recent advances in biotechnology and stem-cell related approaches such as cloning, inner cell mass transfer (ICM), and the stem-cell-associated techniques (SCAT) for the generation of gametes and ultimately embryos of highly endangered species, such as the northern white rhinoceros (Ceratotherium simum cottoni) of which only two female individuals are left. Both, ART and aART greatly depend on and benefit from the rapidly evolving cryopreservation techniques and biobanking not only of genetic, but also of viable cellular materials suitable for the generation of induced pluripotent stem cells (iPSC). The availability of cryopreserved materials bridges gaps in time and space, thereby optimizing the available genetic variability and enhancing the chance to restore viable populations.
... Earlier, rat-mouse [242] , human-mouse [245] , and sheep-goat [246] chimeras were documented. However, blastocyst injection has failed to introduce stem cells into primate embryos [247] . ...
Article
The recent progress in derivation of pluripotent stem cells (PSCs) from farm animals opens new approaches not only for reproduction, genetic engineering, treatment and conservation of these species, but also for screening novel drugs for their efficacy and toxicity, and modelling of human diseases. Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages, or lost their cellular potency; indicating that the protocols which allowed the derivation of murine or human embryonic stem (ES) cells were not sufficient to support the maintenance of ES cells from farm animals. This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support naïve pluripotency in ES cells from livestock species. However, the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging, and requires further refinements. Here, we review the current achievements in the derivation of PSCs from farm animals, and discuss the potential application areas.
... caroli ↔ M. musculus: [8][9][10][11]; rat ↔ mouse: [12,13]), or injection of embryonic stem cells (ESCs) into the blastocysts (mouse ↔ rat: [14]; mouse ↔ rat and rat ↔ mouse: [15][16][17][18]; A. sylvaticus ↔ M. musculus: [19]). There have been also a number of successful attempts to produce live chimeras between two species of domesticated animals belonging to the same gender, such as zebu and cattle [20,21], as well as between species belonging to two different genders, such as sheep and goat [22][23][24][25][26]. It should be strongly stressed, however, that, so far, these sophisticated techniques have always led to the production of the chimeric newborns and have never given rise to a "pure" newborn specifically different from the recipient female. ...
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The full-term development of the xenogeneic embryo in the uterus of the mother of different species is very restricted and can occur only in certain groups of closely related mammals. In the case of mouse↔rat chimeras, the inter-specific uterine barrier is less hostile to inter-specific chimeric fetuses. In current work, we tested the development of mouse and rat fetuses in uteri of females of the opposite species. We created chimeric mouse↔rat blastocysts by injection of mouse embryonic stem cells (ESCs) into rat 8-cell embryos and rat ESCs into 8-cell mouse embryos. Chimeras were transferred to the foster mothers of the opposite species. Despite a huge number of transferred embryos (>1000 in total for both variants), only one live fetus derived solely from the mouse ESCs was isolated at E13.5 from the rat uterus. All other fetuses and newborns were chimeric or were built only from the cells of the recipient embryo. We examined the possible reason for such an outcome and found that the xenogeneic fetuses are eliminated at the perigastrulation stage of development. Thus, we conclude that in the rat↔mouse combination even when extraembryonic tissues of the chimeric embryo are composed solely of the cells of the same species as the female to which embryos are transferred, the full-term development of the pure xenogeneic fetus is very unlikely.
... These fundamental studies indicated the aggregation ability of isolated blastomeres from early cleavage stage embryos to produce chimeric blastocysts that can be transferred to foster mothers (recipients) for the production of intraspecific chimeras. Based on these experiences and with the aim to increase the understanding of reproductive incompatibilities between species, in addition to providing a successful approach for interspecific hybridization, researchers were able to generate sheepgoat chimeras by using two basic techniques: embryo aggregation (Meinecke-Tillmann and Meinecke, 1984;Fehilly et al., 1984b) and embryonic cell injection into host blastocysts (Polzin et al., 1987). These procedures were based on the combination of blastomeres of two species, surrounding blastomeres of two species with each other, or injecting cells of the inner cell mass of one species into the blastocyst cavity of different species. ...
Article
Sheep and goats are valuable livestock species that have been raised for their production of meat, milk, fiber, and other by-products. Due to their suitable size, short gestation period, and abundant secretion of milk, sheep and goats have become important model animals in agricultural, pharmaceutical, and biomedical research. Genome engineering has been widely applied to sheep and goat research. Pronuclear injection and somatic cell nuclear transfer represent the two primary procedures for the generation of genetically modified sheep and goats. Further assisted tools have emerged to enhance the efficiency of genetic modification and to simplify the generation of genetically modified founders. These tools include sperm-mediated gene transfer, viral vectors, RNA interference, recombinases, transposons, and endonucleases. Of these tools, the four classes of site-specific endonucleases (meganucleases, ZFNs, TALENs, and CRISPRs) have attracted wide attention due to their DNA double-strand break-inducing role, which enable desired DNA modifications based on the stimulation of native cellular DNA repair mechanisms. Currently, CRISPR systems dominate the field of genome editing. Gene-edited sheep and goats, generated using these tools, provide valuable models for investigations on gene functions, improving animal breeding, producing pharmaceuticals in milk, improving animal disease resistance, recapitulating human diseases, and providing hosts for the growth of human organs. In addition, more promising derivative tools of CRISPR systems have emerged such as base editors which enable the induction of single-base alterations without any requirements for homology-directed repair or DNA donor. These precise editors are helpful for revealing desirable phenotypes and correcting genetic diseases controlled by single bases. This review highlights the advances of genome engineering in sheep and goats over the past four decades with particular emphasis on the application of CRISPR/Cas9 systems.
... In this case, removal of zona pellucidae from two (in some cases, there are more) preimplanted embryos which will be then aggregated within a single zona [4] . After the in vitro compaction, the chimeric embryos will be transferred to the recipients. ...
... Injection of certain blastomeres of single morula into the inner-cell-mass of an another embryo will result in the production of chimeras that are termed as 'Injection chimeras' [5] . There are only few reports available on the successful production of chimeras in case of sheep, goats and cattle, and the chimeras between sheep and goats are done by aggregating halves or quarters of embryos from both sheep and goat [4] . ...
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Manipulation of embryos, especially micromanipulation includes various microscopic treatments or techniques that help in achieving greater fertilization potential that helps in achieving greater pregnancy rates. Micromanipulation techniques are not really simple, but it requires specialized instruments and equipments. Also, the person associated with micromanipulation techniques should be highly experienced and knowledged. The present review helps in understanding the basic concepts associated with various techniques of micromanipulation.
... It is the case of mice Mus musculus Linnaeus, 1758 and Mus caroli Bonhote, 1902, chicken-quail hybrid and pheasant-turkeys hybrids (Bammi et al. 1966, Benirschke 1967, Rossant et al. 1983). Hybrids stemming from more distant species have reduced fertility or are even sterile as in the crossings mouse -rat and sheep -goat (Polzin et al. 1987, MacLaren et al. 1993. ...
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The Common quail Coturnixcoturnix Linnaeus, 1758 is a wild migratory bird which is distributed in Eurasia and North Africa, everywhere with an accelerating decline in population size. This species is protected by the Bonn and Berne conventions (1979) and by annex II/1 of the Birds Directive (2009). In Algeria, its breeding took place at the hunting centre in the west of the country. Breeding errors caused uncontrolled crosses between the Common quail and Japanese quail Coturnixjaponica Temminck & Schlegel, 1849. In order to help to preserve the natural genetic heritage of the Common quail and to lift the ambiguity among the populations of quail raised in Algeria, it seemed essential to begin to describe the chromosomes of this species in the country since no cytogenetic study has been reported to date. Fibroblast cultures from embryo and adult animal were initiated. Double synchronization with excess thymidine allowed us to obtain high resolution chromosomes blocked at prometaphase stage. The karyotype and the idiogram in GTG morphological banding (G-bands obtained with trypsin and Giemsa) corresponding to larger chromosomes 1–12 and ZW pair were thus established. The diploid set of chromosomes was estimated as 2N=78. Cytogenetic analysis of expected hybrid animals revealed the presence of a genetic introgression and cellular chimerism. This technique is effective in distinguishing the two quail taxa. Furthermore, the comparative chromosomal analysis of the two quails and domestic chicken Gallusgallusdomesticus Linnaeus, 1758 has been conducted. Differences in morphology and/or GTG band motifs were observed on 1, 2, 4, 7, 8 and W chromosomes. Neocentromere occurrence was suggested for Common quail chromosome 1 and Chicken chromosomes 4 and W. Double pericentric inversion was observed on the Common quail chromosome 2 while pericentric inversion hypothesis was proposed for Chicken chromosome 8. A deletion on the short arm of the Common quail chromosome 7 was also found. These results suggest that Common quail would be a chromosomally intermediate species between Chicken and Japanese quail. The appearance of only a few intrachromosomal rearrangements that occurred during evolution suggests that the organization of the genome is highly conserved between these three galliform species.
... Additionally, pigs have a lower cost of maintenance compared with NHPs such as baboon [20]. It is worth noting that the amenability of sheep for interspecies chimera formation has been demonstrated and extensively investigat-ed through studies of sheep-goat interspecies chimeras [21,22]. ...
Article
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The shortage of human organs for transplantation is a devastating medical problem. One way to expand organ supply is to derive functional organs from patient-specific stem cells. Due to their capacity to grow indefinitely in the laboratory and differentiate into any cell type of the human body, patient-specific pluripotent stem (PS) cells harbor the potential to provide an inexhaustible supply of donor cells for transplantation. However, current efforts to generate functional organs from PS cells have so far been unsuccessful. An alternative and promising strategy is to generate human organs inside large animal species through a technique called interspecies blastocyst complementation. In this method, animals comprised of cells from human and animal species are generated by injecting donor human PS cells into animal host embryos. Critical genes for organ development are knocked out by genome editing, allowing donor human PS cells to populate the vacated niche. In principle, this experimental approach will produce a desired organ of human origin inside a host animal. In this mini-review, we focus on recent advances that may bring the promise of blastocyst complementation to clinical practice. While CRISPR/Cas9 has accelerated the creation of transgenic large animals such as pigs and sheep, we propose that further advances in the generation of chimera-competent human PS cells are needed to achieve interspecies blastocyst complementation. It will also be necessary to define the constituents of the species barrier, which inhibits efficient colonization of host animal embryos with human cells. Interspecies blastocyst complementation is a promising approach to help overcome the organ shortage facing the practice of clinical medicine today.
... In the research lab, chimeras are created by introducing cells from one species into the developing embryo or foetus of another. A sheep-goat chimera which was created by inner cell mass transplantation into ovine blastocysts, resulted in the offspring with the head of a goat and the woolly coat of a sheep (Polzin et al., 1987). Hatched chicks with chimeric brains containing cells from both the domestic chicken (Gallus gallusdomesticus) and the Japanese quail (Coturnix coturnixjaponica) (Chicken-quail chimeras) have been produced by transplantation of various regions of the neural tube at the 8-to 15-somite stage (Balaban et al., 1988). ...
Article
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Stem cells have properties of self-renewal and differentiation. Stem cells can be acquired from both embryo and adult body organs. They have also got the ability to differentiate (potency) and to take over the characteristics of any cell type in the body (Plasticity). Because of these efficient characteristics, they can be used to enhance the livestock production by means of chimera production, gene targeting, nuclear transfer, animal cloning, gene transfer and genetic engineering. Also, stem cells can be used to correct various clinical conditions which include cardiac defects, spinal injuries, repairs in tendon, ligament and cartilage and wound healing. This review outlines the properties, origin, classification and plasticity of stem cells. The applications of stem cells in various productive and therapeutic fields are also discussed.
... However, until now there were no publications that describe this approach in sheep. The sheep also has proven to efficiently form an inter-species chimera with goats 23 . ...
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One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.
... Apart from these conventional methods (e.g., aggregation of embryos or embryonic cells derived from the same developmental stage), mixing of embryonic cells from different developmental stages can also result in the chimeric offspring (Gearhart and Oster-Granite, 1981;Nagashima et al., 2004). For example, isolated ICMs are shown to produce chimeras when injected into the blastocoelic cavity of host blastocysts, and at the same time when introduced in 8-cell or morula stage embryos Polzin et al., 1987;Roth et al., 1989;Nagy et al., 1990;Picard et al., 1990). ...
Article
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Stem cells are defined by their capabilities to self-renew and give rise to various types of differentiated cells depending on their potency. They are classified as pluripotent, multipotent, and unipotent as demonstrated through their potential to generate the variety of cell lineages. While pluripotent stem cells may give rise to all types of cells in an organism, Multipotent and Unipotent stem cells remain restricted to the particular tissue or lineages. The potency of these stem cells can be defined by using a number of functional assays along with the evaluation of various molecular markers. These molecular markers include diagnosis of transcriptional, epigenetic, and metabolic states of stem cells. Many reports are defining the particular set of different functional assays, and molecular marker used to demonstrate the developmental states and functional capacities of stem cells. The careful evaluation of all these methods could help in generating standard identifying procedures/markers for them.