RESUMEN
CRISPR-Cas technology has transformed our ability to introduce targeted modifications, allowing unconventional animal models such as pigs to model human diseases and improve its value for food production. The main concern with using the technology is the possibility of introducing unwanted modifications in the genome. In this study, we illustrate a pipeline to comprehensively identify off-targeting events on a global scale in the genome of three different gene-edited pig models. Whole genome sequencing paired with an off-targeting prediction software tool filtered off-targeting events amongst natural variations present in gene-edited pigs. This pipeline confirmed two known off-targeting events in IGH knockout pigs, AR and RBFOX1, and identified other presumably off-targeted loci. Independent validation of the off-targeting events using other gene-edited DNA confirmed two novel off-targeting events in RAG2/IL2RG knockout pig models. This unique strategy offers a novel tool to detect off-targeting events in genetically heterogeneous species after genome editing.
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Sistemas CRISPR-Cas , Edición Génica , Genoma , Animales , Porcinos/genética , Edición Génica/métodos , Técnicas de Inactivación de Genes/métodos , Marcación de Gen/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Secuenciación Completa del Genoma/métodos , Animales Modificados GenéticamenteRESUMEN
COVID-19 impacted abattoirs worldwide. The processing lines became a hotspot for the spread of COVID-19 resulting in plant restructuring and ultimately a critical loss of pig material for research. Commercial sources of pig oocytes are available but are costly and companies were already operating at a maximum capacity for supplying the oocyte needs around the United States. Here, we provide an alternative source of oocytes that are competent to produce live, healthy piglets.
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COVID-19 , Enfermedades de los Porcinos , Femenino , Animales , Porcinos , Ovario , Folículo Ovárico , Oocitos , Recuperación del Oocito/veterinaria , COVID-19/veterinaria , Células del Cúmulo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodosRESUMEN
CRISPR-Cas9 gene editing technology provides a method to generate loss-of-function studies to investigate, in vivo, the specific role of specific genes in regulation of reproduction. With proper design and selection of guide RNAs (gRNA) designed to specifically target genes, CRISPR-Cas9 gene editing allows investigation of factors proposed to regulate biological pathways involved with establishment and maintenance of pregnancy. The advantages and disadvantages of using the current gene editing technology in a large farm species is discussed. CRISPR-Cas9 gene editing of porcine conceptuses has generated new perspectives for the regulation of endometrial function during the establishment of pregnancy. The delicate orchestration of conceptus factors facilitates an endometrial proinflammatory response while regulating maternal immune cell migration and expansion at the implantation site is essential for establishment and maintenance of pregnancy. Recent developments and use of endometrial epithelial "organoids" to study endometrial function in vitro provides a future method to screen and target specific endometrial genes as an alternative to generating a gene edited animal model. With continuing improvements in gene editing technology, future researchers will be able to design studies to enhance our knowledge of mechanisms essential for early development and survival of the conceptus.
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Sistemas CRISPR-Cas , Edición Génica , Embarazo , Femenino , Animales , Porcinos/genética , Edición Génica/métodos , Sistemas CRISPR-Cas/genética , Reproducción/genética , Endometrio/metabolismoRESUMEN
The pig is an ideal model system for studying human development and disease due to its similarities to human anatomy, physiology, size, and genome. Further, advances in CRISPR gene editing have made genetically engineered pigs viable models for the study of human pathologies and congenital anomalies. However, a detailed atlas illustrating pig development is necessary for identifying and modeling developmental defects. Here we describe normal development of the pig abdominal system and show examples of congenital defects that can arise in CRISPR gene edited SAP130 mutant pigs. Normal pigs at different gestational ages from day 20 (D20) to term were examined and the configuration of the abdominal organs was studied using 3D histological reconstructions with episcopic confocal microscopy, magnetic resonance imaging (MRI) and necropsy. This revealed prominent mesonephros, a transient embryonic organ present only during embryogenesis, at D20, while the developing metanephros that will form the permanent kidney are noted at D26. By D64 the mesonephroi are absent and only the metanephroi remain. The formation of the liver and pancreas was observed by D20 and complete by D30 and D35 respectively. The spleen and adrenal glands are first identified at D26 and completed by D42. The developing bowel and the gonads are identified at D20. The bowel appears completely rotated by D42, and testes in the male were descended at D64. This atlas and the methods used are excellent tools for identifying developmental pathologies of the abdominal organs in the pig at different stages of development.
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Edición Génica , Riñón , Abdomen/diagnóstico por imagen , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , Ingeniería Genética , Humanos , Masculino , PorcinosRESUMEN
Genetic modification of animals via selective breeding is the basis for modern agriculture. The current breeding paradigm however has limitations, chief among them is the requirement for the beneficial trait to exist within the population. Desirable alleles in geographically isolated breeds, or breeds selected for a different conformation and commercial application, and more importantly animals from different genera or species cannot be introgressed into the population via selective breeding. Additionally, linkage disequilibrium results in low heritability and necessitates breeding over successive generations to fix a beneficial trait within a population. Given the need to sustainably improve animal production to feed an anticipated 9 billion global population by 2030 against a backdrop of infectious diseases and a looming threat from climate change, there is a pressing need for responsive, precise, and agile breeding strategies. The availability of genome editing tools that allow for the introduction of precise genetic modification at a single nucleotide resolution, while also facilitating large transgene integration in the target population, offers a solution. Concordant with the developments in genomic sequencing approaches, progress among germline editing efforts is expected to reach feverish pace. The current manuscript reviews past and current developments in germline engineering in pigs, and the many advantages they confer for advancing animal agriculture.
RESUMEN
Genetically modified pigs have become valuable tools for generating advances in animal agriculture and human medicine. Importantly, in vitro production and manipulation of embryos is an essential step in the process of creating porcine models. As the in vitro environment is still suboptimal, it is imperative to examine the porcine embryo culture system from several angles to identify methods for improvement. Understanding metabolic characteristics of porcine embryos and considering comparisons with other mammalian species is useful for optimizing culture media formulations. Furthermore, stressors arising from the environment and maternal or paternal factors must be taken into consideration to produce healthy embryos in vitro. In this review, we progress stepwise through in vitro oocyte maturation, fertilization, and embryo culture in pigs to assess the status of current culture systems and address points where improvements can be made.
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Embrión de Mamíferos/fisiología , Creación de Embriones para Investigación/métodos , Porcinos/embriología , Animales , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Fertilización In Vitro , Técnicas de Maduración In Vitro de los OocitosRESUMEN
Background Modeling cardiovascular diseases in mice has provided invaluable insights into the cause of congenital heart disease. However, the small size of the mouse heart has precluded translational studies. Given current high-efficiency gene editing, congenital heart disease modeling in other species is possible. The pig is advantageous given its cardiac anatomy, physiology, and size are similar to human infants. We profiled pig cardiovascular development and generated genetically edited pigs with congenital heart defects. Methods and Results Pig conceptuses and fetuses were collected spanning 7 stages (day 20 to birth at day 115), with at least 3 embryos analyzed per stage. A combination of magnetic resonance imaging and 3-dimensional histological reconstructions with episcopic confocal microscopy were conducted. Gross dissections were performed in late-stage or term fetuses by using sequential segmental analysis of the atrial, ventricular, and arterial segments. At day 20, the heart has looped, forming a common atria and ventricle and an undivided outflow tract. Cardiac morphogenesis progressed rapidly, with atrial and outflow septation evident by day 26 and ventricular septation completed by day 30. The outflow and atrioventricular cushions seen at day 20 undergo remodeling to form mature valves, a process continuing beyond day 42. Genetically edited pigs generated with mutation in chromatin modifier SAP130 exhibited tricuspid dysplasia, with tricuspid atresia associated with early embryonic lethality. Conclusions The major events in pig cardiac morphogenesis are largely complete by day 30. The developmental profile is similar to human and mouse, indicating gene edited pigs may provide new opportunities for preclinical studies focused on outcome improvements for congenital heart disease.
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Cardiopatías Congénitas/embriología , Corazón/embriología , Organogénesis/fisiología , Animales , Modelos Animales de Enfermedad , Imagen por Resonancia Cinemagnética/métodos , Microscopía Confocal , PorcinosRESUMEN
In-vitro maturation (IVM) of oocytes from immature females is widely used in assisted reproductive technologies. Here we illustrate that cumulus cell (CC) expansion, once considered a key indicator of oocyte quality, is not needed for oocytes to mature to the metaphase II (MII) stage and to gain nuclear and cytoplasmic competence to produce offspring. Juvenile pig oocytes were matured in four different media: (1) Basal (-gonadotropins (GN) - FLI); (2) -GN + FLI (supplement of FGF2, LIF, and IGF1); (3) +GN - FLI; and (4) +GN + FLI. There was no difference in maturation to MII or progression to the blastocyst stage after fertilization of oocytes that had been matured in -GN + FLI medium and oocytes matured in +GN + FLI medium. Only slight CC expansion occurred in the two media lacking GN compared with the two where GN was present. The cumulus-oocytes-complexes (COC) matured in +GN + FLI exhibited the greatest expansion. We conclude that FLI has a dual role. It is directly responsible for oocyte competence, a process where GN are not required, and, when GN are present, it has a downstream role in enhancing CC expansion. Our study also shows that elevated phosphorylated MAPK may not be a necessary correlate of oocyte maturation and that the greater utilization of glucose by COC observed in +GN + FLI medium probably plays a more significant role to meet the biosynthetic needs of the CC to expand than to attain oocyte developmental competence. Gene expression analyses have not been informative in providing a mechanism to explain how FLI medium enhances oocyte competence without promoting CC expansion.
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Células del Cúmulo/metabolismo , Fertilización In Vitro/veterinaria , Gonadotropinas/metabolismo , Oocitos/fisiología , Sus scrofa/fisiología , AnimalesRESUMEN
Phenylalanine hydroxylase-deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6-7. The affected pig exhibited hyperphenylalaninemia (2000-5000 µM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.
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Hígado/metabolismo , Fenilalanina Hidroxilasa/genética , Fenilalanina/genética , Fenilcetonurias/genética , Adolescente , Adulto , Animales , Sistemas CRISPR-Cas/genética , Dieta , Modelos Animales de Enfermedad , Edición Génica , Humanos , Hígado/efectos de los fármacos , Fenotipo , Fenilalanina/metabolismo , Fenilalanina/farmacología , Fenilcetonurias/dietoterapia , Fenilcetonurias/metabolismo , Fenilcetonurias/patología , PorcinosRESUMEN
Assisted reproductive technologies in the pig are critical for the production of genetically modified pigs as models of human disease and to improve production agriculture. Methods of oocyte maturation, fertilization, and culture all play an extremely important role in how the embryo, fetus, and offspring will develop. In this chapter, we discuss the historical methods and recent advances that have been essential in promoting efficient and competent embryo development. Here we describe the procedures that can be used to mature, fertilize, and culture pig embryos to the blastocyst stage.
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Blastocisto/metabolismo , Técnicas de Cultivo de Embriones/métodos , Fertilización In Vitro/métodos , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/metabolismo , Porcinos/embriología , Animales , Blastocisto/citología , Desarrollo Embrionario , Oocitos/citologíaRESUMEN
Improper composition of culture medium contributes to reduced viability of in vitro-produced embryos. Glutamine (Gln) is a crucial amino acid for preimplantation embryos as it supports proliferation and is involved in many different biosynthetic pathways. Previous transcriptional profiling revealed several upregulated genes related to Gln transport and metabolism in in vitro-produced porcine blastocysts compared to in vivo-produced counterparts, indicating a potential deficiency in the culture medium. Therefore, the objective of this study was to determine the effects of Gln supplementation on in vitro-produced porcine embryo development, gene expression, and metabolism. Cleaved embryos were selected and cultured in MU2 medium supplemented with 1 mM Gln (control), 3.75 mM Gln (+Gln), 3.75 mM GlutaMAX (+Max), or 3.75 mM alanine (+Ala) until day 6. Embryos cultured with +Gln or +Max had increased development to the blastocyst stage and total number of nuclei compared to the control (P < 0.05). Moreover, expression of misregulated transcripts involved in glutamine and glutamate transport and metabolism was corrected when embryos were cultured with +Gln or +Max. Metabolomics analysis revealed increased production of glutamine and glutamate into the medium by embryos cultured with +Max and increased consumption of leucine by embryos cultured with +Gln or +Max. As an indicator of cellular health, mitochondrial membrane potential was increased when embryos were cultured with +Max which was coincident with decreased apoptosis in these blastocysts. Lastly, two embryo transfers by using embryos cultured with +Max resulted in viable piglets, confirming that this treatment is consistent with in vivo developmental competence.
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Técnicas de Cultivo de Embriones , Desarrollo Embrionario/efectos de los fármacos , Glutamina/farmacología , Leucina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Blastocisto/efectos de los fármacos , Blastocisto/metabolismo , Medios de Cultivo , Transferencia de Embrión , Femenino , Fertilización In Vitro , Expresión Génica/efectos de los fármacos , Ácido Glutámico/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Metabolómica , Embarazo , PorcinosRESUMEN
Genetically engineered pigs are often created with a targeting vector that contains a loxP flanked selectable marker like neomycin. The Cre-loxP recombinase system can be used to remove the selectable marker gene from the resulting offspring or cell line. Here is described a new method to remove a loxP flanked neomycin cassette by direct zygote injection of an mRNA encoding Cre recombinase. The optimal concentration of mRNA was determined to be 10 ng/µL when compared to 2 and 100 ng/µL (P < 0.0001). Development to the blastocyst stage was 14.1% after zygote injection with 10 ng/µL. This method successfully removed the neomycin cassette in 81.9% of injected in vitro derived embryos; which was significantly higher than the control (P < 0.0001). Embryo transfer resulted in the birth of one live piglet with a Cre deleted neomycin cassette. The new method described can be used to efficiently remove selectable markers in genetically engineered animals without the need for long term cell culture and subsequent somatic cell nuclear transfer.
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Ingeniería Genética/métodos , Vectores Genéticos/antagonistas & inhibidores , Integrasas/genética , ARN/administración & dosificación , Animales , Vectores Genéticos/química , Integrasas/efectos de los fármacos , Neomicina/química , ARN/genética , Recombinación Genética , Porcinos , Cigoto/citología , Cigoto/efectos de los fármacosRESUMEN
Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.
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Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , ARN Mensajero/genética , Cigoto/metabolismo , Animales , Animales Modificados Genéticamente/genética , Clonación Molecular/métodos , Microinyecciones , ARN Mensajero/administración & dosificación , Porcinos/genéticaRESUMEN
Somatic cell nuclear transfer is a valuable technique for the generation of genetically engineered animals, however, the efficiency of cloning in mammalian species is low (1-3%). Differentiated somatic cells commonly used in nuclear transfer utilize the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively the metabolism of somatic cells contrasts that of the cells within the early embryos which predominately use glycolysis. Early embryos (prior to implantation) are evidenced to exhibit characteristics of a Warburg Effect (WE)-like metabolism. We hypothesized that pharmacologically driven fibroblast cells can become more blastomere-like and result in improved in vitro embryonic development after SCNT. The goals were to determine if subsequent in vitro embryo development is impacted by (1) cloning pharmacologically treated donor cells pushed to have a WE-like metabolism or (2) culturing non-treated donor clones with pharmaceuticals used to push a WE-like metabolism. Additionally, we investigated early gestational survival of the donor-treated clone embryos. Here we demonstrate that in vitro development of clones is not hindered by pharmacologically treating either the donor cells or the embryos themselves with CPI, PS48, or the combination of these drugs. Furthermore, these experiments demonstrate that early embryos (or at least in vitro produced embryos) have a low proportion of mitochondria which have high membrane potential and treatment with these pharmaceuticals does not further alter the mitochondrial function in early embryos. Lastly, we show that survival in early gestation was not different between clones from pharmacologically induced WE-like donor cells and controls.
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Clonación de Organismos , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Técnicas de Transferencia Nuclear , Animales , Femenino , Embarazo , PorcinosRESUMEN
Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called "FLI medium," improves nuclear maturation of oocytes in cumulus-oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus-oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.
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Animales Modificados Genéticamente , Blastocisto/fisiología , Medios de Cultivo/farmacología , Técnicas de Maduración In Vitro de los Oocitos/métodos , Sus scrofa/genética , Animales , Medios de Cultivo/química , Células del Cúmulo/efectos de los fármacos , Células del Cúmulo/fisiología , Femenino , Fertilización In Vitro , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor Inhibidor de Leucemia/farmacología , Tamaño de la Camada/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Técnicas de Transferencia Nuclear , Oocitos/fisiologíaRESUMEN
Most in vitro culture conditions are less-than-optimal for embryo development. Here, we used a transcriptional-profiling database to identify culture-induced differences in gene expression in porcine blastocysts compared to in vivo-produced counterparts. Genes involved in glycine transport (SLC6A9), glycine metabolism (GLDC, GCSH, DLD, and AMT), and serine metabolism (PSAT1, PSPH, and PHGDH) were differentially expressed. Addition of 10 mM glycine to the culture medium (currently containing 0.1 mM) reduced the abundance of SLC6A9 transcript and increased total cell number, primarily in the trophectoderm lineage (P = 0.003); this was likely by decreasing the percentage of apoptotic nuclei. As serine and glycine can be reversibly metabolized by serine hydroxymethyltransferase 2 (SHMT2), we assessed the abundance of SHMT2 transcript as well as its functional role by inhibiting it with aminomethylphosphonic acid (AMPA), a glycine analog, during in vitro culture. Both AMPA supplementation and elevated glycine decreased the mRNA abundance of SHMT2 and tumor protein p53 (TP53), which is activated in response to cellular stress, compared to controls (P ≤ 0.02). On the other hand, mitochondrial activity of blastocysts, mtDNA copy number, and abundance of mitochondria-related transcripts did not differ between control and 10 mM glycine culture conditions. Despite improvements to these metrics of blastocyst quality, transfer of embryos cultured in 10 mM glycine did not result in pregnancy whereas the transfer of in vitro-produced embryos cultured in control medium yielded live births. Mol. Reprod. Dev. 83: 246-258, 2016. © 2016 The Authors.
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Blastocisto/metabolismo , Transferencia de Embrión , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glicina/farmacología , Animales , Transporte Biológico Activo/efectos de los fármacos , Femenino , Embarazo , PorcinosRESUMEN
Animal models of human diseases are critically necessary for developing an in-depth knowledge of disease development and progression. In addition, animal models are vital to the development of potential treatments or even cures for human diseases. Pigs are exceptional models as their size, physiology, and genetics are closer to that of humans than rodents. In this review, we discuss the use of pigs in human translational research and the evolving technology that has increased the efficiency of genetically engineering pigs. With the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system technology, the cost and time it takes to genetically engineer pigs has markedly decreased. We will also discuss the use of another meganuclease, the transcription activator-like effector nucleases , to produce pigs with severe combined immunodeficiency by developing targeted modifications of the recombination activating gene 2 (RAG2).RAG2mutant pigs may become excellent animals to facilitate the development of xenotransplantation, regenerative medicine, and tumor biology. The use of pig biomedical models is vital for furthering the knowledge of, and for treating human, diseases.
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Sistemas CRISPR-Cas , Desoxirribonucleasas , Ingeniería Genética , Porcinos/genética , Animales , Investigación Biomédica , Proteínas de Unión al ADN , Humanos , Modelos Animales , Proteínas NuclearesRESUMEN
The application of embryo-related technology is dependent on in vitro culture systems. Unfortunately, most culture media are suboptimal and result in developmentally compromised embryos. Since embryo development is partially dependent upon Warburg Effect-like metabolism, our goal was to test the response of embryos treated with compounds that are known to stimulate or enhance this Effect. One such compound is 5-(4-chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48). When added during oocyte maturation, the quality of the resultant embryos was compromised, whereas when added to the culture medium after fertilization, PS48 improved both the percentage of embryos that reach the blastocyst stage and the number of nuclei in those blastocysts. Embryonic PS48 treatment resulted in more phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) in blastocyst-stage embryos as compared to the controls. Further, PS48 could replace bovine serum albumin in embryo culture medium, as demonstrated by high-quality embryos that were developmentally competent. The action of PS48 appears to be via stimulation of phosphoinositide-3 kinase and phosphorylation of AKT, which is consistent with stimulation of the Warburg Effect.
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Medios de Cultivo/química , Técnicas de Cultivo de Embriones/métodos , Desarrollo Embrionario/fisiología , Ácidos Pentanoicos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Porcinos/embriología , Animales , Desarrollo Embrionario/efectos de los fármacos , Técnicas In Vitro , Fosforilación/efectos de los fármacosRESUMEN
Culture systems promote development at rates lower than the in vivo environment. Here, we evaluated the embryo's transcriptome to determine what the embryo needs during development. A previous mRNA sequencing endeavour found upregulation of solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 (SLC7A1), an arginine transporter, in in vitro- compared with in vivo-cultured embryos. In the present study, we added different concentrations of arginine to our culture medium to meet the needs of the porcine embryo. Increasing arginine from 0.12 to 1.69mM improved the number of embryos that developed to the blastocyst stage. These blastocysts also had more total nuclei compared with controls and, specifically, more trophectoderm nuclei. Embryos cultured in 1.69mM arginine had lower SLC7A1 levels and a higher abundance of messages involved with glycolysis (hexokinase 1, hexokinase 2 and glutamic pyruvate transaminase (alanine aminotransferase) 2) and decreased expression of genes involved with blocking the tricarboxylic acid cycle (pyruvate dehydrogenase kinase, isozyme 1) and the pentose phosphate pathway (transaldolase 1). Expression of the protein arginine methyltransferase (PRMT) genes PRMT1, PRMT3 and PRMT5 throughout development was not affected by arginine. However, the dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2 message was found to be differentially regulated through development, and the DDAH2 protein was localised to the nuclei of blastocysts. Arginine has a positive effect on preimplantation development and may be affecting the nitric oxide-DDAH-PRMT axis.
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Amidohidrolasas/metabolismo , Arginina/farmacología , Desarrollo Embrionario/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Óxido Nítrico/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Núcleo Celular/metabolismo , Expresión Génica/efectos de los fármacos , PorcinosRESUMEN
Over the past decade the technology to characterize transcription during embryogenesis has progressed from estimating a single transcript to a reliable description of the entire transcriptome. Northern blots were followed by sequencing ESTs, quantitative real time PCR, cDNA arrays, custom oligo arrays, and more recently, deep sequencing. The amount of information that can be generated is overwhelming. The challenge now is how to glean information from these vast data sets that can be used to understand development and to improve methods for creating and culturing embryos in vitro, and for reducing reproductive loss. The use of ESTs permitted the identification of SPP1 as an oviductal component that could reduce polyspermy. Microarrays identified LDL and NMDA as components to replace BSA in embryo culture media. Deep sequencing implicated arginine, glycine, and folate as components that should be adjusted in our current culture system, and identified a characteristic of embryo metabolism that is similar to cancer and stem cells. Not only will these characterizations aid in improving in vitro production of embryos, but will also be useful for identifying, or creating conditions for donor cells that will be more likely to result in normal development of cloned embryos. The easily found targets have been identified, and now more sophisticated methods are being employed to advance our understanding of embryogenesis. Here the technology to study the global transcriptome is reviewed followed by specific examples of how the technology has been used to understand and improve porcine embryogenesis both in vitro and in vivo.