By employing somatic cell nuclear transfer (SCNT), the cloning of animals from several species has been accomplished. For both food production and biomedical research, pigs stand out as a significant livestock species, closely resembling humans physiologically. During the previous two decades, the cloning of numerous swine breeds has taken place to serve a wide range of purposes, such as those in medicine and farming. A detailed protocol for producing cloned pigs via somatic cell nuclear transfer is presented in this chapter.
Transgenesis, in conjunction with somatic cell nuclear transfer (SCNT) in pigs, opens up promising avenues in biomedical research, particularly for xenotransplantation and disease modeling. Handmade cloning (HMC), a simplified technique for somatic cell nuclear transfer (SCNT), produces cloned embryos in large numbers by circumventing the need for micromanipulators. The porcine-specific adjustments to HMC for both oocytes and embryos have made it uniquely efficient. This efficiency is evident in a blastocyst rate above 40%, 80-90% pregnancy rates, 6-7 healthy offspring per litter, and a drastic reduction in losses and malformations. In conclusion, this chapter illustrates our HMC protocol for the aim of generating cloned pigs.
Differentiated somatic cells acquire totipotency through somatic cell nuclear transfer (SCNT), a technique of substantial importance in developmental biology, biomedical research, and agricultural applications. Transgenesis-mediated rabbit cloning might result in a more effective use of rabbits in mimicking diseases, testing drugs, and producing human proteins for medical purposes. This chapter elucidates the SCNT protocol for the purpose of producing live cloned rabbits.
Somatic cell nuclear transfer (SCNT) technology's utility in animal cloning, gene manipulation, and genomic reprogramming research is undeniable. Despite its efficacy, the standard mouse SCNT protocol still presents a significant financial burden, demands extensive labor, and necessitates substantial hours of dedicated effort. Hence, our efforts have been focused on decreasing the expense and simplifying the mouse SCNT process. The techniques to leverage low-cost mouse strains and the procedures for mouse cloning are examined in detail in this chapter. While this modified SCNT protocol will not elevate the efficiency of mouse cloning, it presents a more economical, straightforward, and less taxing alternative, enabling more experiments and a larger yield of offspring within the same timeframe as the conventional SCNT procedure.
Beginning in 1981, the field of animal transgenesis has undergone consistent advancement, resulting in more efficient, cheaper, and faster methods. A new age of genetically modified organisms is dawning, thanks to advancements in genome editing technologies, particularly CRISPR-Cas9. genetic cluster Synthetic biology, or re-engineering, is what some researchers identify as characterizing this new era. However, the field of high-throughput sequencing, artificial DNA synthesis, and the engineering of artificial genomes is experiencing rapid progress. The improvement of livestock, animal disease modeling, and the production of medical bioproducts is made possible by the symbiotic advancements in animal cloning, using the somatic cell nuclear transfer (SCNT) technique. SCNT's role in genetic engineering is apparent in its capacity to produce animals from genetically modified cells. This chapter considers the rapidly advancing technologies driving this biotechnological revolution and their association with the field of animal cloning.
The process of cloning mammals routinely entails the introduction of somatic nuclei into enucleated oocytes. Cloning is instrumental in maintaining desirable animal characteristics, contributing to germplasm conservation, and is utilized in other beneficial applications as well. A key obstacle to the broader use of this technology lies in its relatively low cloning efficiency, inversely proportional to the differentiation state of the donor cells. New data suggests that adult multipotent stem cells are instrumental in increasing cloning efficiency, while the greater potential of embryonic stem cells in this area remains largely confined to studies on mice. Investigating the derivation of pluripotent or totipotent stem cells from livestock and wild species and their interactions with epigenetic mark modulators in donor cells is likely to lead to increased cloning efficiency.
As essential power plants within eukaryotic cells, mitochondria also serve as a significant biochemical hub. The impairment of mitochondria, possibly due to mutations in the mitochondrial genome (mtDNA), can affect organismal fitness and lead to debilitating human diseases. Rhapontigenin solubility dmso Uniparentally transmitted through the maternal lineage, mtDNA is a multi-copy, highly variable genome. A range of mechanisms within the germline actively combats heteroplasmy, characterized by the co-existence of multiple mitochondrial DNA variants, and inhibits the expansion of mtDNA mutations. Hepatic differentiation However, the reproductive biotechnology of nuclear transfer cloning can alter mtDNA inheritance, creating novel genetic mixes that might be unstable, leading to physiological consequences. This review examines the present comprehension of mitochondrial inheritance, focusing on its transmission pattern in animals and human embryos developed through nuclear transplantation.
The spatial and temporal expression of specific genes is precisely controlled by the intricate cellular process of early cell specification in mammalian preimplantation embryos. For the embryo and placenta to develop correctly, the initial cell segregation of the inner cell mass (ICM) and the trophectoderm (TE) is absolutely necessary. Somatic cell nuclear transfer (SCNT) facilitates the development of a blastocyst comprising both inner cell mass and trophectoderm lineages from a differentiated somatic cell's nucleus, indicating the crucial need to reprogram the differentiated genome into a totipotent state. The efficient generation of blastocysts using somatic cell nuclear transfer (SCNT) contrasts with the often-compromised full-term development of SCNT embryos, a predicament primarily linked to placental malformations. Our review delves into early cell fate decisions within fertilized embryos and then compares them to those observed in SCNT-derived embryos. The intent is to identify any alterations caused by SCNT that may contribute to the comparatively low efficiency of reproductive cloning.
The study of epigenetics examines heritable changes in gene expression and resulting phenotypes, aspects not dictated by the primary DNA sequence. The epigenetic mechanisms primarily involve DNA methylation, histone tail modifications, and non-coding RNA molecules. Epigenetic reprogramming, in mammalian development, manifests in two distinct and sweeping global waves. Gametogenesis marks the occurrence of the first stage, and fertilization is immediately followed by the second. Factors such as exposure to pollutants, improper nutrition, behavioral traits, stress, and the conditions of in vitro cultures can negatively affect the process of epigenetic reprogramming. We detail the key epigenetic processes that occur during the preimplantation stage of mammalian development, such as genomic imprinting and X chromosome inactivation. Furthermore, we delve into the adverse consequences of somatic cell nuclear transfer cloning on epigenetic reprogramming, exploring molecular strategies to mitigate these negative effects.
Somatic cell nuclear transfer (SCNT) into enucleated oocytes effectively restructures the nucleus of lineage-committed cells, restoring their totipotency. SCNT research, culminating in the cloning of amphibian tadpoles, paved the way for the advancement of cloning technology, as breakthroughs in biology and technique allowed cloning of mammals directly from adult animals. Through the use of cloning technology, fundamental biological questions have been addressed, enabling the propagation of desirable genomes and contributing to the creation of transgenic animals or patient-specific stem cells. While not insurmountable, the technical intricacies of somatic cell nuclear transfer (SCNT) and the comparatively low rate of successful cloning still pose a significant hurdle. Nuclear reprogramming encountered hurdles, as revealed by genome-wide techniques, exemplified by persistent epigenetic marks from the originating somatic cells and genome regions resistant to the reprogramming process. Deciphering the rare reprogramming events conducive to full-term cloned development will likely necessitate technological advancements in large-scale SCNT embryo production coupled with comprehensive single-cell multi-omics profiling. SCNT cloning's versatility is undeniable, but ongoing advancements are predicted to sustain and elevate excitement about its diverse applications.
Despite the widespread occurrence of the Chloroflexota phylum, its biology and evolutionary trajectory are poorly understood, primarily due to the limitations of cultivation methods. From the sediments of hot springs, we isolated two motile, thermophilic bacterial strains: these belong to the genus Tepidiforma, a part of the Dehalococcoidia class within the Chloroflexota phylum. Through cultivation experiments using stable carbon isotopes, cryo-electron tomography, and exometabolomics, three remarkable characteristics emerged: flagellar motility, a peptidoglycan-composed cell envelope, and heterotrophic activity related to aromatic and plant-derived compounds. Within the Chloroflexota phylum, flagellar motility is absent outside this genus, and the presence of peptidoglycan in the cell envelopes of Dehalococcoidia has not been confirmed. In cultivated Chloroflexota and Dehalococcoidia, these attributes are atypical; ancestral character reconstructions suggest flagellar motility and peptidoglycan-containing cell envelopes were ancestral in Dehalococcoidia, subsequently lost before a significant diversification into marine ecosystems. While flagellar motility and peptidoglycan biosynthesis demonstrate predominantly vertical evolutionary histories, the evolution of enzymes for degrading aromatics and plant-associated compounds displayed a complex and predominantly horizontal pattern.