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Abstract
Genetically identical nonhuman primates can provide a powerful animal model for gene therapy and research activities where the physiological parameters directly or indirectly under study are heritable. Here we demonstrate that nuclear transfer is a viable technology for the production of identical rhesus macaques. Oocytes recovered from gonadotropin-treated females were enucleated by aspiration of the first polar body and underlying ooplasm, then activated by cycloheximide exposure. Individual diploid blastomeres, recovered from in vitro-fertilization-produced embryos (either fresh or frozen-thawed) and used as nuclear donors, were injected under the zona pellucida of enucleated (chromosome-free) oocytes and fused by electric pulses. The reconstituted embryos were cocultured on buffalo rat liver cells before cryostorage and transfer to synchronized host mothers. Of the 9 females receiving a total of 29 reconstituted embryos, 3 became pregnant, with two live births resulting, one male and one female. The parentage of both infants was established unequivocally by genotype analysis at 7 highly variable short tandem repeat loci.
... Recently, several transgenic swine HD models were also generated [132][133][134] ; these models also showed more obvious neurodegeneration and motor disorders than that of mice with the same length of CAG repeats. Through CRISPR/Cas9-mediated knock-in (KI) on pig fibroblast cells and somatic cell nuclear transfer technique, a HD KI pig model was successfully created [135] . This model expresses 150 CAG repeats under the endogenous HTT promoter, leading to selective neurodegeneration as well as movement disorders, effectively mimicking typical HD pathology and clinic features. ...
... This model expresses 150 CAG repeats under the endogenous HTT promoter, leading to selective neurodegeneration as well as movement disorders, effectively mimicking typical HD pathology and clinic features. Importantly, similar to the transgenic HD monkey, the HD KI pig is also inheritable [135] , making it possible to generate a large number of HD pigs in the future based on the potent reproductive capability of swine. Although the HD KI pig seems to be an ideal model, the macaque monkey model is more suitable for investigating emotional and psychiatric activity. ...
Neurodegenerative diseases (NDs), such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral sclerosis (ALS), are pathologically characterized by progressive loss of selective populations of neurons in the affected brain regions and clinically manifested by cognitive, motor, and psychological dysfunctions. Since aging is the major risk factor for NDs and the elderly population is expected to expand considerably in the coming decades, the prevalence of NDs will significantly increase, leading to a greater medical burden to society and affected families. Despite extensive research on NDs, no effective therapy is available for NDs, largely due to a lack of complete understanding of the pathogenesis of NDs. Although research on small animal and rodent models has provided tremendous knowledge of molecular mechanisms of disease pathogenesis, few translational successes have been reported in clinical trials. In particular, most genetically modified rodent models are unable to recapitulate striking and overt neurodegeneration seen in the patient brains. Non-human primates (NHPs) are the most relevant laboratory animals to humans, and recent studies using NHP neurodegeneration models have uncovered important pathological features of NDs. Here, we review the unique features of NHPs for modeling NDs and new insights into AD, PD, and ALS gained from animal models, highlight the contribution of gene editing techniques to establishing NHP models, and discuss the challenges of investigating NHP models.
... Due to their relatively low reproductive rates, longer generation intervals, and lack of germline integrating and gene-modifiable embryonic stem cells, it is difficult to produce NHP models through endogenous gene KI or knock-out in one-cell embryos, as is practicable in rodents . Although macaque cloning (Meng et al., 1997), especially through somatic cell nuclear transfer (Liu et al., , 2019, has been achieved, the efficiency of this process is very low and needs further improvement. As most genetically based NDs are linked to point mutations, emerging technologies in base editing and prime editing (Yang et al., 2019a;Yeh et al., 2020) may facilitate the generation of better animal models carrying precise human genetic mutations. ...
Neurodegenerative diseases (NDs) are a group of debilitating neurological disorders that primarily affect elderly populations and include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). Currently, there are no therapies available that can delay, stop, or reverse the pathological progression of NDs in clinical settings. As the population ages, NDs are imposing a huge burden on public health systems and affected families. Animal models are important tools for preclinical investigations to understand disease pathogenesis and test potential treatments. While numerous rodent models of NDs have been developed to enhance our understanding of disease mechanisms, the limited success of translating findings from animal models to clinical practice suggests that there is still a need to bridge this translation gap. Old World non-human primates (NHPs), such as rhesus, cynomolgus, and vervet monkeys, are phylogenetically, physiologically, biochemically, and behaviorally most relevant to humans. This is particularly evident in the similarity of the structure and function of their central nervous systems, rendering such species uniquely valuable for neuroscience research. Recently, the development of several genetically modified NHP models of NDs has successfully recapitulated key pathologies and revealed novel mechanisms. This review focuses on the efficacy of NHPs in modeling NDs and the novel pathological insights gained, as well as the challenges associated with the generation of such models and the complexities involved in their subsequent analysis.
... Belajar dari riset Domba Dolly pada tahun 1996, pertama kali di dunia keberhasilan teknik kloning metode transfer nukleus dari kultur sel tepi dapat melahirkan kloning domba (Campbell et al., 1996). Setelahnya, setahun berikutnya riset kloning dengan teknik serupa yang diterapkan pada monyet rhesus berhasil melahirkan monyet rhesus klon (Meng et al., 1997). Metode transfer nukleus (TN) secara langsung terbukti ber kontribusi dalam teknik kloning dalam bioteknologi reproduksi hewan (Simerly et al., 2014). ...
Bab ini membahas pengantar bioteknologi hewan, kloning, transgenik, inseminasi buatan (IB), fertilisasi in-vitro (FIV), transfer embrio (TE), spermatozoa seksing, kriopreservasi, bioteknologi veteriner, pengayaan pakan, keuntungan dan kerugian bioteknologi hewan.
... Belajar dari riset Domba Dolly pada tahun 1996, pertama kali di dunia keberhasilan teknik kloning metode transfer nukleus dari kultur sel tepi dapat melahirkan kloning domba (Campbell et al., 1996). Setelahnya, setahun berikutnya riset kloning dengan teknik serupa yang diterapkan pada monyet rhesus berhasil melahirkan monyet rhesus klon (Meng et al., 1997). Metode transfer nukleus (TN) secara langsung terbukti ber kontribusi dalam teknik kloning dalam bioteknologi reproduksi hewan (Simerly et al., 2014). ...
Buku ini berisikan bahasan tentang bioteknologi dan aplikasinya dalam berbagai bidang kehidupan, teknologi dna rekombinan, teknologi enzim, manfaat mikrobioorganisme sebagai model dan sarana aplikasi bioteknologi, produk-produk bioteknologi yang menggunakan system mikroorganisme, prinsip dasar pemuliaan tanaman dengan teknik kultur jaringan, bioteknologi akuatik, bioteknologi hewan, DNA fingerprinting, bioteknologi medis, pengaturan etik dalam bioteknologi. Bioteknologi telah berkembang pesat khususnya bioteknologi modern. Bioteknologi modern telah mampu melakukan berbagai proses penting dalam dunia industri di beberapa bidang antara lain bidang kesehatan, pangan, pertanian, industri lainnya serta lingkungan. Untuk pembelian dapat mengunjungi tautan https://globaleksekutifteknologi.co.id/bioteknologi/
... Belajar dari riset Domba Dolly pada tahun 1996, pertama kali di dunia keberhasilan teknik kloning metode transfer nukleus dari kultur sel tepi dapat melahirkan kloning domba (Campbell et al., 1996). Setelahnya, setahun berikutnya riset kloning dengan teknik serupa yang diterapkan pada monyet rhesus berhasil melahirkan monyet rhesus klon (Meng et al., 1997). Metode transfer nukleus (TN) secara langsung terbukti ber kontribusi dalam teknik kloning dalam bioteknologi reproduksi hewan (Simerly et al., 2014). ...
... In addition, autism NHP models have been generated by SHANK3 gene knockout via CRISPR-Cas9 [12]. In general, these reported gene editing for the generation of NHP models either by using lentivirus or CRISPR-Cas9 and all needed microinjection on monkey oocytes or zygotes [13][14][15][16], which need a lot of monkeys as oocyte donors and embryo transfer recipients due to low efficiency. ...
Nonhuman primates (NHPs) have been considered as the best models for biomedical research due to their high similarities in genomic, metabolomic, physiological and pathological features to humans. However, generation of genetically modified NHPs through traditional methods, such as microinjection into the pronuclei of one-cell embryos, is prohibitive due to the targeting efficiency and the number of NHPs needed as oocyte/zygote donors. Using spermatogonial stem cells (SSCs) as the target of gene editing, producing gene-edited sperm for fertilization, is proven to be an effective way to establish gene editing animal disease models. In this experiment, we used ultrasound to guide the echo dense injection needle into the rete testis space, allowing the EGFP lentivirus to be slowly injected at positive pressure from the rete testis into seminiferous tubules. We found Thy1 can be used as a surface marker of cynomolgus monkey SSCs, confirming that SSCs carry the GFP gene. Finally, we successfully obtained transgenic sperm, with a similar freezing and recovery rate to that of WT animals.
... Sci. 2023, 10, 17 2 of 10 published their findings, including sheep [7,8]; rabbits [9,10]; pigs [11]; mice [12]; monkeys [13]; goats [14,15]; and camels [16]. These studies opened new horizons for scientists to continue cloning research, such as interspecies and interspecific cloning of Bos gaurus into bovines [17]; various mammal species into bovine [18]; Bos gaurus into bovine [19,20]; Bos javanicus into bovine [21]; bovine-ovine [22]; cat into bovine [23,24]; cattle, goat, and cat into Bubalus bubalis buffalo [25]; cattle and pig [26]; cat into cow [27]; cat into cow [28]; human into bovine [29]; human into rabbit [30], Asiatic cheetah into cat [31]; human into bovine [32]; human into rabbit [33]; argali (Ovis ammon into sheep) [34]. ...
Cloning, commonly referred to as somatic cell nuclear transfer (SCNT), is the technique of enucleating an oocyte and injecting a somatic cell into it. This study was carried out with interspecific SCNT technology to clone the Arabian Oryx utilizing the oryx’s fibroblast cells and transfer it to the enucleated oocytes of a domestic cow. The recipient oocytes were extracted from the cows that had been butchered. Oryx somatic nuclei were introduced into cow oocytes to produce embryonic cells. The study was conducted on three groups, Oryx interspecific somatic cell nuclear transfer into enucleated oocytes of domestic cows, cow SCNT “the same bovine family species”, used as a control group, and in vitro fertilized (IVF) cows to verify all media used in this work. The rates of different embryo developmental stages varied slightly (from 1- cell to morula stage). Additionally, the oryx interspecies Somatic cell nuclear transfer blastocyst developmental rate (9.23%) was comparable to that of cow SCNT (8.33%). While the blastula stage rate of the (IVF) cow embryos exhibited a higher cleavage rate (42%) in the embryo development stage. The results of this study enhanced domestic cow oocytes’ ability to support interspecific SCNT cloned oryx, and generate a viable embryo that can advance to the blastula stage.
... This concept originated from the work of Gurdon and colleagues who were the first to use nuclear transfer to produce frogs with a somatic cell (Gurdon et al., 1958), and, thus, gave rise to the term somatic cell nuclear transfer (SCNT) ( Figure 5). This was followed by the transfer of cultured embryonic-derived (Campbell et al., 1996) and somatic cells to produce the first mammalian offspring, namely Dolly the Sheep (Wilmut et al., 1997); and blastomeres to produce rhesus macaque monkeys (Meng et al., 1997), cattle (Bondioli et al., 1990;Prather et al., 1987), and pigs (Prather et al., 1989). ...
The mitochondrial genome resides in the mitochondria present in nearly all cell types. The porcine (Sus scrofa) mitochondrial genome is circa 16.7 kb in size and exists in the multimeric format in cells. Individual cell types have different numbers of mitochondrial DNA (mtDNA) copy number based on their requirements for ATP produced by oxidative phosphorylation. The oocyte has the largest number of mtDNA of any cell type. During oogenesis, the oocyte sets mtDNA copy number in order that sufficient copies are available to support subsequent developmental events. It also initiates a program of epigenetic patterning that regulates, for example, DNA methylation levels of the nuclear genome. Once fertilized, the nuclear and mitochondrial genomes establish synchrony to ensure that the embryo and fetus can complete each developmental milestone. However, altering the oocyte's mtDNA copy number by mitochondrial supplementation can affect the programming and gene expression profiles of the developing embryo and, in oocytes deficient of mtDNA, it appears to have a positive impact on the embryo development rates and gene expression profiles. Furthermore, mtDNA haplotypes, which define common maternal origins, appear to affect developmental outcomes and certain reproductive traits. Nevertheless, the manipulation of the mitochondrial content of an oocyte might have a developmental advantage.
... Without reviewing the extensive list of pathogens that can be transferred through ART for all species, generally the same disease risks that exist for domestic or laboratory counterparts can be expected for non-domestic spe- Geoffroy cat (Leopardus geoffryi) OHE-GR, PMGR - [106] Golden cat (Catopuma temminckii) OHE-GR, PMGR - [106] Indian desert cat (Felis silvestris ornate) -iET [109,110] Jaguar (Panthera onca) OHE-GR, PMGR - [106] Jaguarundi (Puma yagouaroundi) L-OR SX-iET [110,111] Jungle cat (Felis chaus) OL - [110] Leopard (Panthera pardus) OHE-GR, PMGR - [106] Leopard cat (Prionailurus bengalensis) UL/IVF, iSCNT, OHE-GR, PMGR SX-iET [109,112,113] Lion (Panthera leo) L-OR, OHE-GR, PMGR ET [106,114,115] Ocelot (Leopardus pardalis) -L-ET [102,116] Puma (Felis concolor) L-OR, OHE-GR, PMGR - [106,117] Sand cat (Felis margarita) iSCNT ET [118] Serval (Leptailurus serval) L-OR, OHE-GR, PMGR - [106,119] Snow Leopard (Panthera unica) OHE-GR, PMGR - [106] Tiger (Panthera tigris) L-OR, OHE-GR, PMGR SX-ET [106,109,120,121] Canidae Gray wolf (Canis lupus) iSCNT SX-iET [122] Mexican gray wolf (Canis lupus baileyi) OVX-GR - [123] Silver fox (Vulpes vulpes) -UL, ET [124,125] Ursidae American black bear (Ursus americanus) -UL, SX-ET [126] Panda (Ailuropoda melanoleuca) PMGR - [127] [128,129] Polecat (Mustela putorius) -SX-UL, SX-ET [129] African mastomys (Praomys coucha) IVF ET [130] Primates Baboon (Papio) SX-OR, UL, L-OR SX-ET, ET [115,[131][132][133][134] Chimpanzee (Pan troglodytes) TVOPU - [135,136] Cynomologus monkey (Macaca fascicularis) IVF ET [137,138] Lowland gorilla (Gorilla gorilla gorilla) TVOPU ET [139][140][141][142] Marmoset (Callithrix jacchus) OVX-GR ET, UL [143][144][145][146][147] Rhesus macaque (Macaca mulatta) L-OR L-ET [148][149][150][151][152] Pig tailed macaque (Macaca nemestrina) SX-OR - [153] Marsupialia Bushtail possum (Trichosurus vulpecula) -SX-ET [154] Fat-tailed dunnart (Sminthopsis crassicaudata) -SX-ET [155] Quokka (Setonix brachyurus) SX-ET [156] Wombat (Lasiorhinus latifrons) PMGR - [157] Tammar wallaby (Macropus eugenii) -ET [154,158] disease surveillance program, or from a free-ranging wild animal where the regional disease incidence and health status of the animal are unknown. Risk assessments in zoological institutions should consider whether there are routine or annual health exams; for example, in felid and primate species, and whether preshipment exams are conducted and whether new additions to the collection undergo a quarantine period. ...
... 42 Advances in the use of ARTs in this important species include cryopreservation of sperm; 41 artificial insemination; 34 in vitro fertilization (IVF); 3 and intracytoplasmic sperm injection (ICSI). 30 Establishing effective and reproducible rhesus macaque ARTs is critical in allowing the birth of live offspring after blastomere nuclear transfer, 28 somatic cell nuclear transfer, 10 mitochondrial replacement in oocytes, 43 and autologous grafting of prepubertal rhesus testis, followed by ICSI and embryo development. 16 Moreover, recent advances in gene editing now allow NHP genome manipulation in one cell embryos (zygotes) for the subsequent generation of transgenic models of human disease. ...
Advances in assisted reproductive technologies in rhesus macaques have allowed the development of valuable models of human disease, particularly when combined with recent techniques for gene editing. While the ability to perform in vitro fertilization (IVF) in rhesus macaques is well established, this procedure has not yet been optimized. Specifically, damage to the sperm caused by cryopreservation (cryodamage) may lead to unsuccessful artificial insemination and low fertilization and blastocyst formation rates in vitro. To address this, we systematically assessed 2 cryopreservation methods and 4 recovery methods in the following 3 interdependent experiments: 1) comparing sperm survival after vitrification or slow-freezing; 2) comparing simple wash (SW), density gradient centrifugation (DGC), swim-up (SU), and glass wool filtration (GWF) for removal of cryoprotectants and isolation of motile sperm after thawing; and 3) evaluating the efficacy for IVF of the 2 best methods of isolating thawed sperm. We found that after vitrification, only 1.2 ± 0.3% of thawed sperm were motile, whereas after slow-freezing, 42 ± 5% of thawed sperm were motile. SW was significantly better than all other isolation methods for the recovery of total sperm and for the recovery of sperm with an intact plasma membrane. The isolation methods had no significant differences in the recovery of motile sperm or sperm with progressive motility. However, IVF of ova with sperm recovered by DGC resulted in 5% more embryos and 25% more blastocysts than did IVF with sperm recovered by SW. Although additional studies are required to optimize sperm cryopreservation in rhesus macaques, our study showed that slow-freezing, coupled with DGC, provided the highest efficacy in providing functional sperm for in vitro use.
... This research led to the achievement of mammalian cloning by somatic cell nuclear transfer (SCNT), attained in 1996, with the birth of Dolly, the sheep [3]. This work was soon followed by studies reporting cloning in other mammalian species [4][5][6]. These results revealed that the genome of even fully differentiated cells remains functionally intact and that, under proper conditions, can support the development of a whole organism. ...
BACKGROUND
Conventional cell reprogramming involves converting a somatic cell line into induced pluripotent stem cells (iPSC), which subsequently can be re-differentiated to specific somatic cell types. Alternatively, partial cell reprogramming converts somatic cells into other somatic cell types by transient expression of pluripotency genes thus generating intermediates that retain their original cell identity, but are responsive to appropriate cocktails of specific differentiation factors. Additionally, biological rejuvenation by partial cell reprogramming is an emerging avenue of research.
OBJECTIVE
Here, we will briefly review the emerging information pointing to partial reprogramming as a suitable strategy to achieve cell reprogramming and rejuvenation, bypassing cell dedifferentiation.
METHOD
In this context, regulatable pluripotency gene expression systems are the most widely used at present to implement partial cell reprogramming. For instance, we have constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein and oct4, sox2, klf4 and c-myc genes (known as the Yamanaka genes or OSKM).
RESULTS
Partial cell reprogramming has been used to reprogram fibroblasts to cardiomyocytes, neural progenitors and neural stem cells. Rejuvenation by cyclic partial reprogramming has been achieved both in vivo and in cell culture using transgenic mice and cells expressing the OSKM genes, respectively, controlled by a regulatable promoter.
CONCLUSIONS
Partial reprogramming emerges as a powerful tool for the genesis of iPSC-free induced somatic cells of therapeutic value and for the implementation of in vitro and in vivo rejuvenation keeping cell type identity unchanged.
... However, one notable exception was cloning of non-human primates by SCNT; over the past decade, attempts by multiple laboratories across the world have failed. Rhesus monkeys were first cloned using blastomeres derived from in vitro fertilization-produced embryos as donors for nuclear transfer [6]. In addition, a rhesus macaque named 'Tetra' was created through 'embryo splitting', a process where the cells in an embryo are split at the eight-cell stage to create four identical two-cell embryos [7]. ...
In this chapter, we deal with the change from the exception to the rule in biological systems, both by the action of nature and by the changes that occur due to human action. We talk about the origin of life on planet Earth, the first organisms that colonized primitive environments and changed the atmosphere, giving rise to new forms of life, the appearance of eukaryotic, multicellular organisms, and the different forms of reproduction. We focus on events and changes that were initially considered teratological and that are familiar to our current vision. We also mention adaptations, plasticity, and different phenotypes that became advantages and allowed organisms to continue living in different environments. On the other hand, we point to global processes that affect humans and that in many cases are caused by humans. We discuss examples of diseases that turn into pandemics, the processes of environmental pollution, and accelerated climate change. Finally, we will discuss the changes in scientific ideas, which are closely linked to the social context at each moment in human history, the changes in the different fields of study and within society itself.
Somatic cell nuclear transfer (SCNT) successfully clones cynomolgus monkeys, but the efficiency remains low due to a limited understanding of the reprogramming mechanism. Notably, no rhesus monkey has been cloned through SCNT so far. Our study conducts a comparative analysis of multi-omics datasets, comparing embryos resulting from intracytoplasmic sperm injection (ICSI) with those from SCNT. Our findings reveal a widespread decrease in DNA methylation and the loss of imprinting in maternally imprinted genes within SCNT monkey blastocysts. This loss of imprinting persists in SCNT embryos cultured in-vitro until E17 and in full-term SCNT placentas. Additionally, histological examination of SCNT placentas shows noticeable hyperplasia and calcification. To address these defects, we develop a trophoblast replacement method, ultimately leading to the successful cloning of a healthy male rhesus monkey. These discoveries provide valuable insights into the reprogramming mechanism of monkey SCNT and introduce a promising strategy for primate cloning.
The term 'cloning' refers to the production of genetically identical individuals but has meant different things throughout the history of science: a natural means of reproduction in bacteria, a routine procedure in horticulture, and an ever-evolving gamut of molecular technologies in vertebrates. Mammalian cloning can be achieved through embryo splitting, somatic cell nuclear transfer, and most recently, by the use of induced pluripotent stem cells. Several emerging biotechnologies also facilitate the propagation of genomes from one generation to the next whilst bypassing the conventional reproductive processes. In this review, we examine the state of the art of available cloning technologies and their progress in species other than humans and rodent models, in order to provide a critical overview of their readiness and relevance for application in endangered animal conservation.
Stem cells have self-renewal capability and can proliferate and differentiate into a variety of functionally active cells that can serve in various tissues and organs. This review discusses the history, definition, and classification of stem cells. Human pluripotent stem cells (hPSCs) mainly include embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Embryonic stem cells are derived from the inner cell mass of the embryo. Induced pluripotent stem cells are derived from reprogramming somatic cells. Pluripotent stem cells have the ability to differentiate into cells derived from all three germ layers (endoderm, mesoderm, and ectoderm). Adult stem cells can be multipotent or unipotent and can produce tissue-specific terminally differentiated cells. Stem cells can be used in cell therapy to replace and regenerate damaged tissues or organs.
The cloning between different animals known as interspecific somatic cell nuclear transfer (iSCNT) was carried out for endangered species. The iSCNT has been characterized by a poor success rate due to several factors that influence the formation of the SCNT in various cytoplasms. The cell cycle of the transferred somatic cell, the passage number of the cultured somatic cell, the mitochondria oocytes, and their capabilities are among these factors. This study investigates the role of the passage number of the Arabian Oryx somatic cell culture when transplanted to an enucleated domestic cow oocyte and embryo development in vitro. The fibroblast somatic cell of the Arabian Oryx was cultured for several passage lanes (3–13). The optimal passage cell number was found to be 10–13 Oryx cell lines that progressed to various cell stages up to the blastula stage. There was some variation between the different passage numbers of the oryx cell line. The 3–9 cell line did not show a good developmental stage. These could be attributed to several factors that control the iSCNT as stated by several investigators. More investigation is needed to clarify the role of factors that affect the success rate for the iSCNT.
Genetic modification of nonhuman primate (NHP) zygotes is a useful method for the development of NHP models of human diseases. This review summarizes the recent advances in the development of assisted reproductive and genetic manipulation techniques in NHP, providing the basis for the generation of genetically modified NHP disease models. In this study, we review assisted reproductive techniques, including ovarian stimulation, in vitro maturation of oocytes, in vitro fertilization, embryo culture, embryo transfer, and intracytoplasmic sperm injection protocols in marmosets. Furthermore, we review genetic manipulation techniques, including transgenic strategies, target gene knock-out and knock-in using gene editing protocols, and newly developed gene-editing approaches that may potentially impact the production of genetically manipulated NHP models. We further discuss the progress of assisted reproductive and genetic manipulation techniques in NHP; future prospects on genetically modified NHP models for biomedical research are also highlighted.
Transferring nuclear material between mammalian oocytes or embryos from unrelated individuals offers an opportunity to exchange not only nuclear genetic materials but also their cytoplasm. The cytoplasm of a mammalian oocyte contains large amounts of mitochondria/mitochondrial (mt) DNA, which is maternally inherited. Given this unique feature of mitochondrial inheritance, it is feasible to replace mitochondria/mtDNA with donor cytoplasm if a reliable technology allowed for efficient transfer of only the nuclear material in eggs or in early embryos. Such a technology is deemed mitochondrial replacement therapy (MRT). MRT encompasses the potential utilization for germline gene therapy and for a theory for future assisted conception, which would overcome recurrent in vitro fertolization failure due to impaired cytoplasmic function (i.e., reproductive aging). In this chapter, the mitochondrial biogenesis in gametogenesis, during fertilization, early embryonic development, and mitochondrial disease; the research milestones and limitation of MRT; and MRT-derived potential application in future assisted conception were summarized.
Somatic cell nuclear transfer (SCNT) is an important technique for biological science research. Cytoplasm injection cloning technology (CICT) was developed to improve the reprogramming efficiency as well as to overcome the limitations of SCNT. CICT uses an additional cytoplasm fused with an enucleated oocyte to restore the cytoplasmic volume of the cloned embryo, and this method could improve the reprogramming efficiency of the cloned embryo. In this study, we show that CICT can be adapted to mouse species to overcome the inefficiency of the SCNT method. In this study, results indicate that the two-cell embryo and blastocyst rates of cloned embryos with the use of the CICT method were significantly higher (p < 0.05) than that of the SCNT method (96.6% ± 1.1% vs. 86.7% ± 6.0%, 29.5% ± 2.6% vs. 22.1% ± 3.0%, respectively). Furthermore, the apoptotic cell number per blastocyst was significantly lower in the CICT group than that in the SCNT group (1.7 ± 0.2 vs. 2.9 ± 0.3, p < 0.05). Moreover, the acH3K9/K14 expression level in the CICT group was greater than that of the SCNT group (p < 0.05), and the relative acH3K56 level in the CICT group was significantly (p < 0.05) higher than that in the SCNT group. These results indicate that CICT helps improve the in vitro developmental competence and quality of cloned embryos.
Production of nonhuman primate (NHP) embryos in vitro begins with recovery of gametes. Females undergo a controlled ovary stimulation to produce multiple preovulatory follicles from which oocytes may be recovered. Mature ova are subjected to in vitro fertilization (IVF) and presumptive zygotes are cultured to the intended stage of development. Essential to this practice is the culture medium unique to each step in the process. Here we describe medium preparation, oocyte recovery, in vitro fertilization, and in vitro culture (IVC) of embryos in the rhesus macaque model.
Research with non‐human primates (NHP) has been essential and effective in increasing our ability to find cures for a large number of diseases that cause human suffering and death. Extending the availability and use of genetic engineering techniques to NHP will allow the creation and study of NHP models of human disease, as well as broaden our understanding of neural circuits in the primate brain. With the recent development of efficient genetic engineering techniques that can be used for NHP, there's increased hope that NHP will significantly accelerate our understanding of the etiology of human neurological and neuropsychiatric disorders. In this article, we review the present state of genetic engineering tools used in NHP, from the early efforts to induce exogeneous gene expression in macaques and marmosets, to the latest results in producing germline transmission of different transgenes and the establishment of knockout lines of specific genes. We conclude with future perspectives on the further development and employment of these tools to generate genetically engineered NHP. (i) Non‐human primates (NHP) models are essential in biomedical research; (ii) genetically engineered NHP are essential for human genetic disorders; (iii) this article reviews the present state of genetic engineering in NHP. Approaches to generate genetically engineered non‐human primates.
Microinjection/micromanipulation is more than 100 years old. It is a technique that is instrumental in biomedical research and healthcare. Its longevity lies in its preciseness in mechanical retrieval, or delivery of biological materials, which in some cases is simply necessary or more effective than other retrieval/delivery means. Microinjection is favored for its straightforwardness in transferring contents from micromolecules to macromolecules and from organelles to cells. Microinjection/micromanipulation has been practiced over the century like an art form. Variations in handlings and instruments can be tolerated to a surprising degree with satisfactory outcomes. Throughout the century, microinjection developed as an indispensable tool along with the evolution of biomedical fields: from transgenics to gene targeting, from animal cloning to human infertility treatment, from nuclease-guided genetic engineering to RNA-guided genome editing (Fig. 1). The birth of the CRISPRology rejuvenated microinjection. For microinjection/micromanipulation, the second century has already begun with the early arrival of computerized instrumentation and lately of the high-throughput nanomanipulators potentially operable by artificial intelligence. As we yin-yang both systemic and precision approaches in research and medicine, microinjection will no doubt continue to find its unique place in the future.
Non-human primates (NHPs) are similar with humans in their anatomy, physiology, genetics and neural functions, and in their cognition, emotions and social behavior. The NHP model has played an important role in the biomedical research, development of vaccines and drugs and new therapies for many diseases. Recent advances in genome editing have generated NHP models for human diseases. Genome editing integrated with derivation of NHP pluripotent stem cells now provides an optimal resource to regenerate medicine. © The Author(s) 2017. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. All rights reserved.
The viral cause of AIDS was not established until 1983 (BARRE-SINNOUSSI et al. 1983; GALLO et al. 1983; LEVY et al. 1984), although HIV could have existed in the human population for 50–200 years previously (LI et al. 1988; SMITH et al. 1988; Yokoyama et al. 1988) and may have found its way to Europe and North America as early the 1960s (CORBITT et al. 1990; GARRY et al. 1988; HUMINER et al. 1987). The epidemic continues to sweep across the world, making its most recent inroads in Asia (MANN and TARANTOLA 1996). Vaccines to prevent other viral diseases have been highly successful (PLOTKIN and MORTIMER 1994), and enthusiasm for obtaining one to contain AIDS, once HIV was identified as its cause, was high (RUSSELL 1984). Others pointed out apparently daunting obstacles (ADA 1988; COHN and STEINMAN 1988; FERDINAND et al. 1987; LEVY 1988; NATHANSON and GONZALES-SCARANO 1989), and the intervening years have seen swings of optimism and pessimism about a timetable for eventual success.
Embryonic stem cells (ESC) hold promise for the treatment of human medical conditions but are allogeneic. Here, we consider the differences between autologous pluripotent stem cells produced by nuclear transfer (NT-ESCs) and transcription factor-mediated, induced pluripotent stem cells (iPSCs) that impact the desirability of each of these cell types for clinical use. The derivation of NT-ESCs is more cumbersome and requires donor oocytes; however, the use of oocyte cytoplasm as the source of reprogramming factors is linked to a key advantage of NT-ESCs - the ability to replace mutant mitochondrial DNA (mtDNA) in a patient cell (due to either age or inherited disease) with healthy donor mitochondria from an oocyte. Moreover, in epigenomic and transcriptomic comparisons between isogenic iPSCs and NT-ESCs, the latter produced cells that more closely resemble bona fide ESCs derived from in fertilized embryos. Thus, although NT-ESCs are more difficult to generate than iPSCs, the ability of somatic cell nuclear transfer to replace aged or diseased mitochondria and the closer epigenomic and transcriptomic similarity between NT-ESCs and bona fide ESCs may make NT-ESCs superior for future applications in regenerative medicine. This article is protected by copyright. All rights reserved.
This chapter reviews the ongoing research efforts to derive genetically identical nonhuman primates. The genetic similarity between nonhuman primates and humans makes nonhuman primates, especially the rhesus monkey, a uniquely suited model for biomedical research. Because of these similarities, nonhuman primates are not only ideally suited for research of traditional human maladies but also offer a unique system for developing and testing new therapeutic approaches in human medicine. Therapeutic applications of gene therapy; cell, tissue, and organ transplantation; stem cell therapies; and vaccines and drugs for unique primate conditions can be reliably studied only in nonhuman primates. Conditions specific to primates include asthma, hepatitis, leishmaniasis, and parainfluenza. During the past few years, ultrasonography for the imaging of ovaries has been employed successfully, which has improved the evaluation of stimulation protocols. The size of the ovaries and the number and size of developing follicles are measured to evaluate the response, which has resulted not only in improved yields of good quality oocytes but also in a reduction of the number of animals undergoing surgical recovery attempts in the absence of a satisfactory ovarian stimulation. However, superovulation yields inconsistent and unpredictable numbers of good quality oocytes, where the average yield from a first-time superovulated animal ranges between 0 and 156, averaging between 15 and 20 per collection. The development of nonhuman primate models for human disease through nuclear transplantation or embryo splitting can greatly benefit ongoing research on drug testing, vaccine development, gene therapy, and many other areas of importance. Genetically identical animals would allow the use of a fewer numbers of animals in the research while dramatically increasing the accuracy and interpretation of results due to reduced or eliminated genetic variability between animals.
The embryo preimplantation genetic diagnosis (PID) was proposed to avoid the birth of genetically abnormal babies without the recourse to medical abortion as it may occur with prenatal diagnosis (PND). In fact the number, availability and statute of the 'potential persons' submitted to diagnosis by either PID or PND are not comparable. It is why the medical indications for PID already begin to overstep 'particularly serious diseases'. However PID will be limited to few cases till the supervention of certain new biological procedures in addition to molecular genetics. These procedures include: (1) increasing production of mature oocytes; (2) use of numerous genetic tests for each resulting embryo; and (3) cellular cloning of 'the best' available embryo. By cryopreserving many copies of this elected embryo one can assure the birth of a corresponding child despite the low pregnancy rate from each embryo transfer. To prevent such a eugenic future scenario, an international specific regulation on PID is a matter of urgency. We propose to limit DNA identification in ex vivo embryos to only one gene, whereas chromosome aneuploidies should be detected without restriction.
Mice can be cloned from cultured and noncultured adult-, fetus-, male-, or female-derived cells. The efficiency is typically 1–2%, with an average of one to two offspring being produced for every 100 starting one-cell embryos. Cloned embryos usually undergo developmental arrest prior to or soon after implantation, and offspring often develop cloning-associated phenotypes. This can be the result of various fundamental mechanisms in biology, including cancer and both cellular and organismal aging. Notwithstanding the relative ease of mouse embryo culture and manipulation, mouse metaphase II (MII) oocytes are so sensitive that a conventional microinjection through pipette tips wider than 2 ∝m is impracticable and generally results in cell lysis. The intractability of microinjecting mouse metaphase II oocytes through such wider pipettes was eventually overcome with the application of piezo-actuated micromanipulation. Cloning by transfer of somatic cell nuclei from adult mice was finally achieved using a single transfer step in 1997, marking the beginning of a new chapter in mouse cloning. In this development, mouse NT biology was brought alongside that of the larger species. The ability of nucleus donor cells to support development is influenced by the mouse strain from which the cells originate. Cloning by NT from cumulus cells or embryonic stem (ES) cells of hybrid strains is significantly more efficient than analogous NT from inbred strains. The most efficient cloning was achieved when enucleated oocytes of hybrid mice were injected with cumulus cell nuclei of hybrid mice.
The generation of animals from somatic cells by nuclear transfer has taught us that cellular differentiation is reversible and not caused by permanent changes to the genome. Rather, the same genetic information is used to establish different gene expression states and thereby cell types with different physiological functions. Experimental manipulations of gene expression states by nuclear transplantation can provide insights into how cells alter or stably maintain a particular state. An important determinant of the successful transition from a somatic to an embryonic state is the cell cycle stage of oocytes or zygotes at the time of somatic cell nuclear transfer. At interphase, the factors constituting the gene expression program in oocytes or zygotes are associated with chromatin, while in mitosis they localize to the cytoplasm. This physical separation of the genetic information from the major determinants that regulate its expression allows the exchange of the embryonic with a somatic genome, while retaining an embryonic gene expression program. However, embryonic factors can only mediate an efficient transition from somatic to embryonic gene expression if the somatic nucleus is remodeled during cell division and reformed as a nucleus of embryonic morphology at the first interphase after transfer. This suggests that nuclear structure and gene expression are functionally interdependent and require coordinated establishment in a cell type-specific manner during the exit from meiosis or mitosis.
Biological model systems that support descriptive pathology based on gene-environment interactions and span the spectrum from pathogenesis to clinical phenotype are required for basic scientific and clinically meaningful study of developmental disabilities. Developmental disorders encompass a broad range of genetic abnormalities that are subject to epigenetic and environmental modification. Simple nonmammalian organism and rodent model systems have proven extremely useful for the study of gene function related to developmental disability. However, for some developmental disorders, nonprimate orthologs of the affected human gene(s) may not exist. Furthermore, sensory modalities and functionally integrated physiological systems that respond to and are affected by epigenetic and environmental influences are known to be quite different in primate than in nonprimate species. Given these differences, nonprimate models of developmental disabilities may exhibit vastly different phenotypes compared with the human condition. Assisted reproductive technologies (ARTs) encompass an enormous array of skills from both basic sciences and clinical medicine, span the spectrum of most species used for scientific experimentation, and cover molecular to integrated systems approaches to reproductive biology. Advances in embryology and stem cell-based. ARTs are directly responsible for the generation of genetically modified and genetically identical mammals and have had a large impact on progress in rodent models of developmental disability. Thus development and application of ARTs in nonhuman primates represents a promising avenue for expanded use of nonhuman primate models of developmental disabilities.
Putian Black pigs, an improved pig breed in China, are on the edge of extinction and urgently require protection. To establish a method of electro-fusion/activation for reconstructed embryos obtained by Somatic Cell Nuclear Transfer (SCNT), the applicability of microelectrode fusion in electro-fusion/activation of reconstructed embryos from SCNT and its optimal parameters were explored using pig oocyte maturation in vitro for 42-44 h as receptors and ear fibroblast as donors. Researchers also compared microelectrode fusion with traditional chamber fusion and the effects of different culture media (PZM-3 and NCSU-23) on reconstructed embryos were evaluated. Researchers found that the optimal parameters for microelectrode fusion/activation were 16 V, 20 usee and 1 DC in which the fusion efficiency was significantly higher than that of chamber-fusion (84.8 vs. 64.4%) but the rates of embryo cleavage and blastocyst formation showed no statistical difference (p>0.05). The cleavage rates of embryos cultured in PZM-3 or NCSU-23 showed no statistical difference (p>0.05) while the rate of blastocyst formation in PZM-3 was higher than that in NC SU-23 (13.8 vs. 7.84%, p<0.05). These results show that microelectrode fusion/activation can be used in SCNT to obtain cloned embryos from Putian Black pigs and its fusion efficiency is higher than that of traditional chamber fusion. In addition, reconstructed embryo development is optimal in PZM-3. This study establishes a technical basis for SCNT to obtain cloned embryos from Putian Black pigs. Moreover, it can be applied to related fields such as the production of transgenic cloned embryos from Putian Black pigs.
Arrest of cells in the G0/G1 cell cycle phase is desired for somatic cell nuclear transfer procedures. This study determined the arresting effects of 0-2% Dimethyl Sulfoxide (DMSO) for 4-24 h on cell cycle stages of Maccaca fascicularis ear-derived fibroblasts. The effects of DMSO on apoptosis of these cells was also investigated. Cells were obtained from the ear of a 4 years old male monkey. Analysis of cell cycle distribution by fluorescence-activated cell sorting showed that 67.52, 14.37 and 18.11% of normally cycling cells were at G0/G1, S, G2/M phases, respectively. In the groups with 4 h DMSO treatments, cell cycle synchronization in G0/G1 phase for treatment with 1.5 and 2.0% DMSO (82.86 and 86.31 %) was significantly higher than in other groups (0.5% for 78.84% and 1.0% for 78.65% ) or the control group (67.52%). With 24 h treatments, the proportion of cells in G0/G1 was higher with 1 % (90.45%), 1.5% (91.57%) and 2.0% DMSO (98.68%) than with 0.5% DMSO (78.22%) or the control group (70.33%). Under normal culture conditions, 4.63% of cells underwent apoptosis. Treatment with 1.5% DMSO, 2% DMSO for 4 h and 1 % DMSO, 1.5% DMSO, 2% DMSO for 24 h resulted in apoptosis in 10.52, 12.75, 10.42, 12.75 and 17.07% of cells, respectively. In conclusion, the use of DMSO is suitable for cell cycle synchronization because it arrests cells at the G0/G1 phase but it also induces a high level of apoptosis, especially after 24 h for cultures treated with 2% DMSO.
Animal production systems can be broadly classified into three categories: grazing, mixed crop-livestock, and intensive. Originally, all livestock production was grassland based and fell into the grazing category. Grassland systems effectively convert human-inedible materials to high-quality human food. Where climatic, soil, and disease conditions permitted, grassland-based systems developed into mixed crop-livestock systems. On a global basis, mixed crop-livestock systems involve the largest number of animals, generate the most total production, and serve the largest number of people (Seré and Steinfeld 1996). Intensive animal agriculture systems developed more recently, mostly in the vicinity of urban centers when urbanization and income exceeded certain levels. Intensive livestock production is the fastest growing category as a result of several factors, including declining real prices for feed grains, and improved feed conversion ratios (unit feed per unit animal product), animal health, and reproductive rates (Naylor et al. 2005). That animals of most species produce more product per animal in less time when fed nutrient-dense grain diets is one of the factors favoring the growth of intensive systems. Large-scale intensive operations, in which animals are raised in confinement, now account for three-quarters of the global poultry supply, 40 percent of the pork supply, and more than two-thirds of all eggs (Bruinsma 2003). Intensive livestock production systems have dramatically reduced the amount of land needed to produce a unit of animal product, such as a gallon of milk or a pound of meat. For example, over the last century, advances in the genetics, nutrition, and management of U.S. dairy cows have resulted in more than a fourfold increase in milk production per cow, and a threefold improvement in productive efficiency (milk output per feed resource input; VandeHaar and St-Pierre 2006). However, the environmental and ethical sustainability of these intensive production systems is coming increasingly under scrutiny. Large-scale animal operations concentrate environmental pollutants and result in ecological disturbances, and consumers in some countries are increasingly concerned about the health and well-being of animals raised in concentrated animal production systems.
About 99.9% of vertebrate species reproduce sexually. This makes the exceptional 0.1%-the asexual or clonal reproducers-fascinating in their own right, and also uniquely instructive about the biological significance of alternative reproductive modes. This book describes the genetics, ecology, natural history, and evolution of all of the world's approximately 100 "species" of vertebrate animal that routinely display one form or another of clonal or quasi-clonal reproduction. The book investigates the astounding realm of sexual abstinence, from the levels of DNA molecules and somatic cells to whole animals and natural populations. Also described is how scientists have learned to mimic and extend nature's own clonal processes by engineering perfect copies of genes, genomes, and whole animals in the laboratory. By considering the many facets of sexual abstinence and clonal reproduction in vertebrate animals, new light is also shed on the biological meaning and ramifications of standard sexuality.
The promise of nuclear transfer (NT) in producing patient-specific embryonic stem cells for regenerative medicine holds great interest for the treatment of human diseases, whether as tools for "disease in a dish" discoveries or as replacement cells in patient therapies. With the discovery of induced pluripotent stem cells, current debates flourish over whether human NT is necessary, given the limitations of burdensome technical, ethical, and legal issues. Non-human primates (NHP) are superior models for understanding the stepwise events of successful primate NT and could provide easier extrapolations to events in humans than rodent cloning model. Early challenges to producing cloned NHP blastocysts using traditional NT technologies successful in rodent and cows were overcome by modifications to enucleation, artificial activation, and embryo culture in monkeys. Regardless, adoption of these technical advances in human NT has yet to produce stable human pluripotent embryonic stem cells, and recent reports suggest that human meiotic spindle removal as the first step in the cloning process remains detrimental to producing viable human ESC lines. This chapter provides a review on challenges in nuclear transfer in primates - non-human and human alike.
This chapter provides a comprehensive review of the present state of somatic cell nuclear transfer (SCNT)-based cloning, including potential areas of application, with emphasis on the epigenetic reprogramming of the transferred somatic cell nucleus. Common somatic cloning protocols involve several technical steps such as collection and enucleation of the recipient oocyte, preparation and subzonal transfer of the donor cell, fusion of the two components, activation of the reconstructed complex, temporary culture of the reconstructed embryo, and transfer to a foster mother or storage in liquid nitrogen. Basic epigenetic mechanisms include DNA methylation that plays a crucial role in suppressing the activities of parasitic promoters and is thus part of the gene-silencing system in eukaryotic cells. Methylation is associated with silencing of a given gene, but an increasing number of genes are found to be activated by methylation, particularly tumor-suppressor gene. Imprinting represents a specific function of DNA methylation. A typical feature of genomic imprinting is that the two alleles of a given gene are expressed differently. Usually one allele, either the maternal or the paternal, is silenced throughout development by covalent addition of methyl groups to cytosine residues in CpG dinucleotides. DNA demethylation is a first step in epigenetic reprogramming and is essential for Oct4 transcription. The failure of demethylation is associated with impaired development in cloned mice embryos.
To evaluate the effects of ablation of steroid production and the role of testosterone and dihydrotestosterone in the development of secondary follicles of nonhuman primates (Macaca mulatta) in 3 D matrix.
Previous reports have indicated that failure in cloning monkey is attributed to the removal of nuclear mitotic apparatus (NuMA) during enucleation and subsequent abnormal organization of mitotic apparatus. This study investigated the transformation and assembly of tubulin and NuMA protein during the first cell cycle of cloned monkey embryos reconstructed by using enucleated rabbit oocytes as recipients. After the oocyte fused with a fibroblast, extensive microtubule organization was observed around the introduced nucleus in most reconstructed embryos, suggesting the introduction of a somatic cell centrosome. A high proportion of fibroblast nuclei transferred into non-activated oocytes underwent premature chromosome condensation (PCC), transient spindle organization and chromosomes separation, followed by the formation of two pronucleus-like structures. In contrast, fibroblast nuclei in pre-activated ooplasm rarely underwent PCC, but formed a swollen pronucleus-like structure. Normal spindles were observed in about one third of the cloned embryos reconstructed by both methods. After transferring monkey fibroblasts into NuMA-removed enucleated rabbit oocytes, NuMA was localized in pseudo-pronuclei and gradually moved to mitotic spindle poles at the first mitotic spindle poles. NuMA antibody microinjection resulted in spindle disorganization and chromosome misalignment, but did not significantly affect early cleavage. Our findings indicate that: 1. NuMA in donor monkey fibroblast may contribute to form a normal spindle in enucleated rabbit oocyte; 2. when non-activated cytoplasts and pre-activated cytoplasts are used as recipients, the donor nuclei undergo different morphological changes, but yield similar early embryo development; 3. although abnormal spindle organization and chromosome alignment may cause low efficiency of animal cloning, these abnormalities do not significantly affect early cleavage.
The nonhuman primate is a relevant model for human disease that can be used for diverse biomedical investigations. The ability to propagate a founder animal by application of assisted reproductive technologies is pressing, but an even greater need in many studies is access to genetically identical animals. In an effort to create genetically identical monkeys, we evaluated two approaches to monozygotic twinning; blastomere separation, and blastocyst bisection. Embryos were produced by intracytoplasmic sperm injection of oocytes recovered following controlled ovarian stimulation. The quality of demiembryos produced in these efforts was evaluated by quantitating the efficiency of creating identical pairs for embryo transfer, by morphological assessment, by the allocation of cells to the inner cell mass (ICM) and trophectoderm (TE) in the blastocyst, and by the outcome of embryo transfer to synchronized host animals. Pairs were produced in high yield (85%–95%) by both twinning methods. Demiembryos resulting from blastomere separations at the 2- or 4-cell stage grew to blastocysts at the control frequency. Demiblastocysts contained, on average, half the number of cells of the intact controls while maintaining the same ICM:TE or ICM:total cell ratio. The equivalency of demiblastocysts within a set was also evaluated by differential cell counting. Embryo transfers of identical sets led to a 33% clinical pregnancy rate, with two twin pregnancies initiated. Neither pregnancy resulted in term birth of monozygotic twins, but our results are sufficiently encouraging to justify a large-scale twinning trial in the rhesus macaque.
Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean ± SEM, 81 ± 5%, 88 ± 7%, and 87 ± 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 ± 15%) or ICSI control embryos (46 ± 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.
The transfer of nuclei from one cell to another provides a powerful tool for studying the interactions between the cytoplasm of one cell and the nucleus of another. This study was designed to examine the ability of the bovine metaphase oocyte cytoplasm to support mitotic cell cycles under the direction of differentiated somatic cell nuclei of various mammalian species. Skin fibroblast cells from cows, sheep, pigs, monkeys, and rats were used as sources of donor nuclei. Nuclear transfer units produced by fusion of enucleated bovine oocytes and individual fibroblasts from all species examined underwent transition to interphase accompanied by nuclear swelling, further progression through the cell cycle, and completion of the first mitosis. Regardless of the species of donor fibroblasts used, some cleaving units progressed further and developed to advanced stages, as evidenced by continuation of cell proliferation and formation of a blastocoele cavity at the time appropriate for the donor fibroblast species. Although no pregnancies have been carried to term after transfer of embryos into surrogate animals, these observations suggest that mechanisms regulating early embryonic development may be conserved among mammalian species and that bovine oocyte cytoplasm can support the introduced differentiated nucleus regardless of chromosome number, species, or age of the donor fibroblast.
One of the great strengths of the mouse model is the wide array of genetic tools that have been developed. Striking examples include methods for directed modification of the genome, and for regulated expression or inactivation of genes. Within neuroscience, it is now routine to express reporter genes, neuronal activity indicators , and opsins in specific neuronal types in the mouse. However, there are considerable anatomical, physiological, cognitive, and behavioral differences between the mouse and the human that, in some areas of inquiry, limit the degree to which insights derived from the mouse can be applied to understanding human neurobiology. Several recent advances have now brought into reach the goal of applying these tools to understanding the primate brain. Here we describe these advances, consider their potential to advance our understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward.
Harvesting stem cells is inevitably associated with destruction of early embryos. There have been continuing challenges to devaluate the moral status of early embryos. The distinction between preembryo and embryo was mainly based on the lack of individuation. The term preembryo had been introduced by a frog embryologist and then was literally spread around the world because of policy reasons. Thus the definition of preembryo is not yet complete and the term has not yet been used in most medical textbooks including textbooks of human embryology. Preembryo is a period during human development and therefore should be regarded as valuable as an early form of human life. Obtaining embryonic stem cells using SCNT (Somatic Cell Nuclear Transfer) is often called therapeutic cloning as opposed to reproductive cloning that produces human beings by SCNT. Therapeutic cloning used same SCNT to produce human embryos, and therefore it is essentially a process of human cloning. Human cloning is prohibited in most of the world due to ethical issues. Cloning primates including humans using SCNT has not been successful, however, recently there has been a remarkable progress with the help of improved technology of therapeutic cloning. Other countries also recognize the enormous potentials of cloned embryonic stem cells, but no country other than Korea and the UK has allowed therapeutic cloning for fear of the "slippery slope" towards human reproductive cloning. Ongoing researches of therapeutic cloning should be monitored carefully and a broad consensus is needed before allowing further therapeutic cloning. Eventually more ethical ways of obtaining embryonic stem cells should be developed.
Reconstituted sheep embryos have been produced by electrofusion-mediated nuclear transplantation in which single cells derived from embryos at the 16-cell (Day 4) and the inner cell mass (ICM) of early blastocyst stage (Day 6) were fused to unfertilized enucleated secondary oocytes. Electrofusion rates were higher with 16-cell blastomeres (82%) than with ICM cells (47%) and when an alternating current (a.c.) preceded the direct current (d.c.) fusing pulse (88% vs. 47%). The addition of cytochalasin B (7.5% micrograms/ml) to the medium for 1 h after electrofusion significantly improved the development to morula-blastocyst stage of reconstituted embryos derived from both 16-cell blastomeres (11% vs. 35%) and ICM cells (0% vs. 56%), indicating that the cytoskeletal mechanisms operating at the time of oocyte activation may affect the developmental potential of exogenous nuclei. Transplantation of 22 reconstituted morulae and blastocysts from both groups into the uteri of recipient ewes led to the development of four lambs (18%) with the phenotype of the nuclear donor breed. These findings indicate that at least some nuclei derived from transcriptionally active embryos are totipotent and able to be reprogrammed to support full-term development when fused to enucleated secondary oocytes.
Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)
Nuclear transplantation in the mouse embryo was achieved by using a method that combines microsurgical removal of the zygote pronuclei with the introduction of a donor nucleus by a virus-mediated cell fusion technique. Survival of embryos was greater than 90 per cent in tests of this procedure. The embryos developed to term at a frequency not significantly different from that of nonmanipulated control embryos. Because nuclei and cytoplasm from genetically distinct inbred mouse strains can be efficiently interchanged, this procedure may be useful in characterizing possible cytoplasmic contributions to the embryonic and adult phenotype.
This study was an examination of the developmental potential of in vitro fertilization (IVF)-produced rhesus monkey embryos that were cultured in medium alone or cocultured with various cell types. End points were the quality and yield of embryos attaining the expanded or hatched blastocyst stage. A total of 96 IVF-produced embryos were cryopreserved and thawed, and 90 embryos were considered intact and suitable for culture. These embryos were placed into one of five treatment groups consisting of four different cell supports and medium alone. Two primary cultures (bovine oviductal cells [bOVID] and bovine cumulus cells [bCUM]) and two established cell lines (Vero cells and buffalo rat liver cells [BRL]) were utilized for coculture of embryos. Embryos were cultured for up to 14 days, and growth curves were established for all embryos that expanded and/or hatched. The developmental rate for embryos classified as viable varied substantially; in number of days to reach a given stage, early morulae ranged from Days 3 to 9 post-insemination, morulae from Days 4 to 9, blastocysts from Days 6 to 11, expanded blastocysts from Days 7 to 12, and hatched blastocysts from Days 9 to 15. On the basis of developmental curves, 30% of the embryos were arrested upon thawing or shortly after. Of the remaining embryos classified as viable, developmental efficiencies to the hatched blastocyst stage for the various treatments were 1) bOVID, 33%; 2) bCUM, 15%; 3) Vero cells, 9%; 4) BRL, 45%; and 5) medium alone, 8%.(ABSTRACT TRUNCATED AT 250 WORDS)
Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81) that are characteristic of human embryonal carcinoma cells. R278.5 cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers but differentiate or die in the absence of fibroblasts, despite the presence of recombinant human leukemia inhibitory factor. R278.5 cells allowed to differentiate in vitro secrete bioactive chorionic gonadotropin into the medium, express chorionic gonadotropin alpha- and beta-subunit mRNAs, and express alpha-fetoprotein mRNA, indicating trophoblast and endoderm differentiation. When injected into severe combined immunodeficient mice, R278.5 cells consistently differentiate into derivatives of all three embryonic germ layers. These results define R278.5 cells as an embryonic stem cell line, to our knowledge, the first to be derived from any primate species.
Ultraviolet (UV) irradiation in combination with the bisbenzimide stain Hoechst 33342 has been used to visualize the chromosomes before removal of oocytes to be used as cytoplasmic hosts for embryo nuclear transplantation. Short term effects of UV irradiation on the cytoplasmic viability of bovine oocytes matured in vitro were assessed by performing membrane and intracellular studies at 2 and 20 h after exposure to UV irradiation for 0, 30 or 60 s. At the membrane level, loss of integrity was shown by increased lysis and increased retention of the fluorescein diacetate dye in oocytes exposed to 60 s of UV irradiation and uptake of methionine was higher in both irradiated groups. At the intracellular level, methionine incorporation into protein was 5.8 times higher in controls than in oocytes exposed to UV irradiation for 60 s and there was a marked difference in the pattern of protein synthesis. Some changes in protein synthesis were also found in oocytes after 30 s exposure. Moreover, high levels of fluorescence with the dye rhodamine 123 at 20 h after exposure indicated large increases in mitochondrial membrane potential in both groups of UV-irradiated oocytes. Together, these findings indicate that exposure to UV irradiation for periods as short as 60 s causes alterations to both membrane and intracellular components of bovine oocytes matured in vitro. It is concluded that care must be taken when using this methodology to visualize or destroy metaphase chromosomes during enucleation in the embryo cloning protocol.
We previously demonstrated, in luteinizing hormone (LH)-deficient macaques, that follicular growth and maturation occurred with administration of exogenous (recombinant human) follicle stimulating hormone (r-hFSH) alone, and that the oocytes recovered fertilized at a notably higher rate than their counterparts from animals receiving both r-hFSH and r-hLH (Zelinski-Wooten et al., 1995). Here, the developmental potential of embryos produced from animals treated with r-hFSH alone or in combination with r-hLH was evaluated. Embryos (n = 127) were cryopreserved, thawed and either co-cultured on buffalo rat liver cells until the hatched blastocyst stage or transferred to synchronized recipients. Although embryos from each treatment group demonstrated a similar ability to develop to hatched blastocysts with a definitive inner cell mass, a significant difference was seen in cryosurvival (56 versus 78%) and in developmental rate to the hatched blastocyst (12 versus 10 days) between embryos from the r-hFSH alone and the combination group respectively. Pregnancies resulted following oviductal embryo transfers in both groups, with corpus luteum rescue occurring on days 12-16 of the luteal phase. In summary, r-hFSH alone during the pre-ovulatory interval is adequate for the gametogenic events required to produce embryos that develop either in vitro or in vivo; however, exposure to r-hLH may improve embryo viability and the rate of development.
Exciting new opportunities in embryo cloning have been made possible by recent studies on the interaction of the donor nucleus with the recipient cytoplasm after embryo reconstruction. This article reviews information regarding the co-ordination of nuclear and cytoplasmic events during embryo reconstruction, in particular the direct and indirect effects of maturation/ meiosis/mitosis-promoting factor (MPF), upon the transferred nucleus. This will be discussed in relation to DNA replication, the maintenance of correct ploidy, the occurrence of chromosomal abnormalities and development of reconstructed embryos. Although this review is primarily concerned with the reconstruction of mammalian embryos, specific examples from amphibians will also be cited.
The objective of this book is to review the impact of genetic variation on risk of human disease at the different major levels of organization: cells, individuals, families, and populations. The volume begins with a discussion of sources and rates of mutation which ultimately give rise to the vast amount of extant genetic variation. This is followed by presentations of current understanding of how genetic variation is maintained within and among populations. The volume ends with discussions of the implications of such variation for understanding the evolution of our species. This collection gives an unusually broad treatment of the subject, with chapters from some of the leading workers in the field. James Neel’s chapter on human consanguinity effects and M. Otake’s on the genetic effects of radiation associated with the dropping of the Hiroshima and Nagasaki atomic bombs should be singled out for special emphasis. As an up-to-date overview of ongoing research, this work will be of interest to a wide range of workers in the fields of human population genetics, evolution, and epidemiology.
We investigate the contribution of the Iberian bat fauna to the cryptic diversity in Europe using mitochondrial (cytb and ND1) and nuclear (RAG2) DNA sequences. For each of the 28 bat species known for Iberia, samples covering a wide geographic range within Spain were compared to samples from the rest of Europe. In this general screening, almost 20% of the Iberian species showed important mitochondrial discontinuities (K2P distance values > 5%) either within the Iberian or between Iberian and other European samples. Within Eptesicus serotinus and Myotis nattereri, levels of genetic divergence between lineages exceeded 16%, indicating that these taxa represent a complex of several biological species. Other well-differentiated lineages (K2P distances between 5–10%) appeared within Hypsugo savii, Pipistrellus kuhlii and Plecotus auritus, suggesting the existence of further cryptic diversity. Most unsuspected lineages seem restricted to Iberia, although two have crossed the Pyrenees to reach, at leas...
This chapter discusses the progress of nuclear transplantation studies in mammals, the important biological factors influencing the development of eggs and embryos reconstituted by nuclear transplantation, and the prospects for future studies in this field. The transplantation of a nucleus to an appropriate cytoplast and the subsequent analysis of its embryonic development provide the most vigorous and direct tests of genomic totipotency of a cell nucleus. The most advanced studies are those carried out in amphibia. Totipotency, the capacity to direct the formation of fertile frogs, has been demonstrated for the nuclei of many kinds of embryonic cells; pluripotency, as judged by the development of heart-beating larvae, has been proved for all the cell nuclei tested to date.
Micromanipulation and electrofusion were utilized for nuclear transfer in bovine embryos. Embryonic blastomeres from 5-day (estrus = day 0), 6-day, frozen-thawed 5-day, and first-generation nuclear transfer embryos (embryos were themselves a product of nuclear transfer with the original donor being a 5-day embryo) were transferred into bisected bovine oocytes by electrofusion. The percentage of donor cells fusing with the recipient oocytes was compared between different types of donor embryos. The percentage of embryos developing normally into morula or blastocysts following 6 days culture in the sheep oviduct was also recorded and compared between different donor embryo types. No significant differences were found between donor blastomeres for the percent successfully fused to oocytes: 5-day, 294 of 513 (57.3%); 6-day, 252 of 405 (62.2%); frozen-thawed 5-day, 111 of 144 (77.1%); nuclear transfer, 142 of 223 (63.7%); or the percent developing normally following nuclear transfer: 5-day, 92 of 444 (20.7%); 6-day, 84 of 357 (23.5%); frozen-thawed 5-day, 32 of 127 (25.2%); nuclear transfer, 31 of 199 (15.6%). These data suggest that a variety of donor embryos can successfully be utilized for bovine embryo cloning. Also, development of blastomeres from frozen-thawed 5-day donors and from donors that are themselves the product of nuclear transfer suggest that the production of multiple identical offspring is possible by frozen storage of seed stock and serial recloning.
Electroejaculation is an accepted method of semen collection from nonhuman primates. Although both penile and rectal probe stimulation techniques have been used, there has been a general lack of consistency and detail regarding their application. This report describes the collection, processing, and evaluation of rhesus monkey semen contrasting two methods of penile electroejaculation: 1) a constant-voltage method where stimulus current is a variable and 2) a constant-current method where stimulus current is operator-controlled. The constant-current method was the more efficient procedure, requiring a lower stimulus current for successful electroejaculation. The influence on semen quality of potentially toxic agents used in the procedure, surgical glove powder and electrolyte cream, was tested; both were detrimental as measured by motility loss. No correlation was found between coagula volume and sperm numbers. The intra- and interanimal variability in semen samples from six monkeys was also evaluated. Penile electroejaculation, combined with control of stimulus current, provides a consistent, successful, and humane method for the collection of semen in the rhesus monkey.
Fertilization in vitro of rhesus oocytes has been performed in order to provide a model for the study of primate embryonic development. Rhesus monkeys wee superstimulated by daily injections of pregnant mare's serum gonadotropin (PMSG) for 12 days beginning Days 3-5 of menses. An injection of human chorionic gonadotropin (hCG) was given 24 h after the last PMSG injection to induce oocyte maturation. Laparoscopic aspiration of 106 follicles approximately 30 h after hCG injection yielded 87 oocytes (82% recovery). Fifty-three of these oocytes (62%) were enclosed in loose (expanded) cumulus oophorus. Washed, ejaculated rhesus spermatozoa (20 x 106/ml) were preincubated for 4-6 h in a modified Tyrode's solution, then diluted (1/10) into 100-μl culture drops containing oocytes, and caffeine and dibutyryl cAMP (1 mM each). In later experiments, these chemicals were added to the sperm suspensions during the last 1.5 h of preincubation, so that they became diluted 1/10 (final concentrations 0.1 mM each) in the fertilization drops. Sperm and oocytes were coincubated for 18-24 h, then oocytes were either fixed or transferred to fresh culture medium. In 9 experiments a total of 70 rhesus oocytes were inseminated in vitro. Total results for 5 experiments were 13/30 oocytes (43%) showing signs consistent with fertilization (sperm in vitellus and/or more than 1 pronucleus or early stages of cleavage). Four more experiments yielded confirmatory evidence of fertilization. When oocytes were inseminated with live sperm that had been preincubated with caffeine and dibutyryl cAMP, 23/29 oocytes (79%) cleaved at least once; 20 of these developed to the 8-cell stage or further. A further 11 oocytes, randomly selected from the pool of available eggs in the 4 experiments, were inseminated under identical conditions with killed sperm. None of these oocytes showed activation, cleavage or fragmentation during culture for 48-73 h. Activation of oocytes and cleavage resulted only from interaction with live sperm, indicating that fertilization was in progress or had been completed.
This research was undertaken to improve development of parthenogenetic embryos following various combined treatments of ethanol and cycloheximide. In Experiment 1 in vitro matured oocytes (IVM, 24 hr) were treated with 7% ethanol for 5 min followed by incubation in 10 micrograms/ml cycloheximide in Medium 199 for 0 (control), 5, 10, and 20 hr. Development to 2-8 cells following culture for 3 days was similar among treated groups (32-41%; P > 0.05), which was higher than that of controls (6%; P < 0.05). Experiment 2 compared pre-ethanol exposures for 0, 1, 2.5, and 5 min, followed by 5 hr cycloheximide treatment on activation development. One- to 5-min groups resulted in 42-44% cleavage contrasted to 1-12% for controls (P < 0.05). Experiment 3 examined the effect on oocyte development of ethanol and different concentrations of cycloheximide (0, 1, 5, and 10 micrograms/ml). Cleavage to 2-8 cells was similar among the 5 and 10 micrograms/ml cycloheximide groups (36% and 42%, P > 0.05) but lower (P < 0.05) for the 1 micrograms/ml group (24%) and the controls (2-13%). When 5 micrograms/ml cycloheximide was used (Experiment 4), pre-exposure to ethanol (1, 2.5, and 5 min) resulted in more oocytes cleaved (38-41%) than in the cycloheximide alone group (0%) or the control (0%, P < 0.05). Experiment 5 tested blastocyst development of the activated oocytes with or without cytochalasin B treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days.
The present study investigated the decay of maturation-promoting factor (MPF) activity in electrically activated in vitro-matured bovine oocytes and examined the influence of the cell cycle stage of both the donor nucleus and the recipient cytoplasm upon the morphology and DNA synthesis potential of the donor nucleus in reconstructed embryos. The decay of MPF activity was studied both biochemically in electrically activated in vitro-matured oocytes and by morphological examination of nuclear structure in reconstructed bovine embryos. As measured by H1 kinase activity in groups of 10 oocytes, the level of MPF declined rapidly to 30 +/- 4% (of the maximum level in unactivated control oocytes) at 60 min and reached a basal level of 20 +/- 6% at 120 min. This level of activity was then maintained until at least 9 h postactivation. In contrast, when MPF activity was assayed by morphological examination of nuclei in individual reconstructed embryos, the decline in activity occurred over a period of 9 h postactivation. DNA synthesis of nuclei arrested at the G1/S border and in G2 phases of the cell cycle was examined in embryos reconstructed at the time of oocyte activation, i.e., when MPF levels were maximal, and by fusion 10 h postactivation, when no MPF activity could be detected. All nuclei transferred at the time of oocyte activation underwent nuclear envelope breakdown (NEBD) and subsequent DNA synthesis. However, when nuclei were transferred 10 h after activation, no NEBD was observed in any nuclei. Nuclei arrested at the G1/S border or nuclei in S phase when transferred in the absence of NEBD underwent DNA synthesis, while no DNA synthesis was observed in G2 nuclei transferred into this cytoplasmic environment. The results presented show that all nuclei, regardless of cell cycle stage, undergo DNA replication when transplanted into metaphase II (MeII) cytoplasts in which MPF activity is high. From these observations we would suggest that one factor that may contribute to the present low frequency of development of bovine nuclear transfer embryos is the ploidy of the reconstructed embryo after the first cell cycle. In order to maintain correct ploidy of the reconstructed embryo, only G1 nuclei should be transferred at the time of activation, when MPF levels are high. In contrast, when the integrity of the nuclear membrane is maintained by transfer at 10 h postactivation, when MPF activity is absent, the rereplication of G2 nuclei is prevented and correct ploidy of the reconstructed embryo may be maintained.
Hematological traits are commonly assessed markers of health status that have been used in a large number of anthropological studies, especially those focusing on high-altitude adaptation. Despite the wealth of literature on environment-associated variation in these traits, relatively few studies have dealt with the underlying genetic components of hematological measures. The purpose of this study is to estimate heritabilities for eight hematological traits using data obtained from a large pedigreed chimpanzee colony. Seven of the eight hematological traits exhibited significant heritabilities, ranging from h2 = 0.308 for mean cell volume to h2 = 0.834 for red blood cell count. The use of multiple measures per individual proved to be essential for the accurate estimation of heritabilities. We conclude that the underlying genetic variation in hematological traits should be considered when these measures are used in study protocols.
Nuclear transplantations into metaphase II (MII) and S phase oocyte cytoplasm were performed to investigate the influence of recipient cell cycle stage on nuclear function and development of bovine nuclear transplant (NT) embryos. Rate of inactivation of histone H1 kinase and duration of DNA synthesis in activated oocytes were determined. The proportion of S phase blastomeres in in vivo produced day 5.5 bovine embryos was measured. DNA synthesis was also assessed in NT embryos after transfer into MII and S phase cytoplasm. MII NT embryos were produced by fusing a blastomere into a MII oocyte; the fusion pulse served to activate the oocyte. S NT embryos were produced by fusing a blastomere into an early S phase oocyte electrically activated 4 h prior to fusion. Nuclear envelope structure, chromosome constitution, and extent of development were examined in MII and S NT embryos. Histone H1 kinase activity dropped to baseline within 2 h of electrical activation. A second electrical pulse did not alter H1 kinase activity when delivered 4 h after the first pulse. The frequency of S phase blastomeres in day 5.5 bovine embryos ranged from 79% to 100%, depending on the duration of culture in 3H-thymidine. Nuclear transplantation into MII cytoplasm resulted in a transient drop in DNA synthesis over 3.5 h. DNA synthesis resumed at 4.5 h post activation (hpa), concomittantly with initiation of DNA replication in activated oocytes. In contrast, DNA synthesis was not interrupted after transfer into S phase cytoplasm. DNA synthesis persisted until 13.5 hpa, as in activated oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Both follicle stimulating hormone (FSH) and luteinizing hormone (LH) are proposed requirements for follicular growth and steroidogenesis; however, the role of LH in primate folliculogenesis is unclear. Follicular stimulation by recombinant human FSH (n = 5) with and without recombinant LH (1:1; n = 6) following 90 days of gonadotrophin-releasing hormone (GnRH) antagonist (Antide) treatment in macaques was evaluated. Human chorionic gonadotrophin (HCG) was administered when six follicles > or = 4 mm were observed. Oocytes were aspirated 27 h later and inseminated in vitro. Chronic Antide reduced serum oestradiol and bioactive LH to concentrations observed in hypophysectomized rhesus monkeys. Multiple follicular growth required a longer interval following recombinant FSH (12 +/- 1 days) than recombinant FSH+recombinant LH (9 +/- 0.2 days), but the total number of follicles/animal did not differ between groups. The day prior to HCG, oestradiol concentrations were 4-fold less following recombinant FSH compared to recombinant FSH+recombinant LH. With recombinant FSH, more oocytes completed meiosis to metaphase II (51%) and fertilized (89 +/- 5%) relative to recombinant FSH+recombinant LH (12 and 52 +/- 11% respectively). Follicular growth and maturation in LH-deficient macaques occurred with FSH alone. Thus, LH is not required for folliculogenesis in primates. Higher fertilization rates following follicular stimulation with FSH alone suggest that the presence of LH with FSH (1:1) during the pre-ovulatory interval impairs gametogenic events in the periovulatory period.
Nuclear transfer has been used in mammals as both a valuable tool in embryological studies and as a method for the multiplication of 'elite' embryos. Offspring have only been reported when early embryos, or embryo-derived cells during primary culture, were used as nuclear donors. Here we provide the first report, to our knowledge, of live mammalian offspring following nuclear transfer from an established cell line. Lambs were born after cells derived from sheep embryos, which had been cultured for 6 to 13 passages, were induced to quiesce by serum starvation before transfer of their nuclei into enucleated oocytes. Induction of quiescence in the donor cells may modify the donor chromatin structure to help nuclear reprogramming and allow development. This approach will provide the same powerful opportunities for analysis and modification of gene function in livestock species that are available in the mouse through the use of embryonic stem cells.
Multifactorial diseases such as coronary heart disease, cancer, and osteoporosis are of increasing public health concern. Such diseases have a complex etiopathogenesis involving genetic and environmental factors and interactions between these factors. Nonhuman primates are uniquely suited as animal models for complex human diseases because of their close evolutionary relationship to humans. We present the rationale for using nonhuman primates as animal models in research designed to assess the genetic determinants of multifactorial diseases. Strategies and current approaches for the use of nonhuman primate models in genetic research on complex diseases are reviewed.
Several media, some augmented with amino acids, have been formulated recently, based on simplex optimization, to support the preimplantation development of mouse embryos. For the highly limited studies on preimplantation development of nonhuman primate embryos, a complex medium (CMRL-1066) has been employed. Our objective was to compare the developmental ability of rhesus monkey embryos in a simple medium containing amino acids, KSOM/AA, with the complex media used previously. Zygotes (99) were recovered following in vitro fertilization (IVF) from six monkeys, allocated to either CMRL or KSOM/AA both containing 10% fetal calf serum (FCS), and monitored daily until reaching the expanded or hatched blastocyst stage. The distribution of cells between the inner cell mass (ICM) and trophectoderm was determined at the end of culture by differential nuclear staining. Although a greater number of embryos cultured in KSOM/AA vs. CMRL developed to the morula stage (80%) and beyond (66% to expanded blastocyst), the differences were not significant. Such embryos in KSOM/AA did, however, develop at a significantly faster rate, on average, reaching the expanded blastocyst stage 26 hr earlier than did embryos cultured in CMRL. KSOM/AA embryos hatched in less time and had a higher percentage (43 vs. 34) of cells allocated to the ICM. These results indicate that a simple medium, KSOM/AA, in the presence of serum, supports the development of rhesus monkey embryos at high efficiency and at a faster rate than that observed for embryos cultured in the complex medium, CMRL-1066.
Fertilization of mammalian eggs is followed by successive cell divisions
and progressive differentiation, first into the early embryo and
subsequently into all of the cell types that make up the adult animal.
Transfer of a single nucleus at a specific stage of development, to an
enucleated unfertilized egg, provided an opportunity to investigate
whether cellular differentiation to that stage involved irreversible
genetic modification. The first offspring to develop from a
differentiated cell were born after nuclear transfer from an
embryo-derived cell line that had been induced to become
quiescent1. Using the same procedure, we now report the birth
of live lambs from three new cell populations established from adult
mammary gland, fetus and embryo. The fact that a lamb was derived from
an adult cell confirms that differentiation of that cell did not involve
the irreversible modification of genetic material required for
development to term. The birth of lambs from differentiated fetal and
adult cells also reinforces previous speculation1,2 that by
inducing donor cells to become quiescent it will be possible to obtain
normal development from a wide variety of differentiated cells.
Wolf DR Use of recombinant human gonadotropins for repeated follicular stimulation in rhesus monkeys. In: Program of the XVIth Congress of the International Primatological Society and XIXth Conference of American Society of Primatologists
- Mb Zelinski-Wooten
- M Alexander
- Ta Molskness
- Rl Stouffer
Zelinski-Wooten MB, Alexander M, Molskness TA, Stouffer RL,
Wolf DR Use of recombinant human gonadotropins for repeated follicular stimulation in rhesus monkeys. In: Program of the XVIth Congress of the International Primatological Society and XIXth Conference of American Society of Primatologists; 1996; Madison, WI. Abstract 133.
Successful application of PCR-amplified DNA markers for paternity determination in rhesus monkeys (Macaca mulatta) and chimpanzees (Pan troglodytes )
- Jj Ely
- Ml Campbell
- Dl Gonzalez
- Wh Stone
Ely JJ, Campbell ML, Gonzalez DL, Stone WH. Successful application of PCR-amplified DNA markers for paternity determination in
rhesus monkeys (Macaca mulatta) and chimpanzees (Pan troglodytes ). Lab Primate Newsl 1996; 35:1-4.
Program of the XVIth Congress of the International Primatological Society and XIXth Conference of American Society of Primatologists
- M B Zelinski-Wooten
- Alexander M Molskness
- T A Stouffer
Zelinski-Wooten MB, Alexander M, Molskness TA, Stouffer RL,
Wolf DR Use of recombinant human gonadotropins for repeated follicular stimulation in rhesus monkeys. In: Program of the XVIth Congress of the International Primatological Society and XIXth Conference of American Society of Primatologists; 1996; Madison, WI. Abstract 133.
Successful application of PCR-amplified DNA markers for paternity determination in rhesus monkeys (Macaca mulatta) and chimpanzees (Pan troglodytes)
- J J Ely
- M L Campbell
- D L Gonzalez
- W H Stone
Ely JJ, Campbell ML, Gonzalez DL, Stone WH. Successful application of PCR-amplified DNA markers for paternity determination in
rhesus monkeys (Macaca mulatta) and chimpanzees (Pan troglodytes). Lab Primate Newsl 1996; 35:1-4.