RESUMEN
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.
Asunto(s)
Células del Cúmulo/metabolismo , Fertilización In Vitro/veterinaria , Gonadotropinas/metabolismo , Oocitos/fisiología , Sus scrofa/fisiología , AnimalesRESUMEN
In vitro embryo production systems are limited by their inability to consistently produce embryos with the competency to develop to the blastocyst stage, survive cryopreservation, and establish a pregnancy. Previous work identified a combination of three cytokines [fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF1)], called FLI, that we hypothesize improve preimplantation development of bovine embryos in vitro. To test this hypothesis, FLI was supplemented into oocyte maturation or embryo culture medium. Embryos were produced in vitro using abattoir-derived oocytes and fertilized with sperm from a single bull known to have high fertility. After an 18-20 h fertilization period, putative zygotes were cultured in synthetic oviductal fluid (SOF) for 8 days. The addition of FLI to the oocyte maturation medium increased (P < 0.05) the dissociation of transzonal projections at 12, 18, and 24 h of maturation, as well as, the proportion of oocytes that reached the metaphase II stage of meiosis. Additionally, lipid content was decreased (P < 0.05) in the blastocyst stage embryo. The addition of FLI during the culture period increased development to the blastocyst stage, cytoskeleton integrity, and survival following slow freezing, as well as, decreased post thaw cell apoptosis (P < 0.05). In conclusion, the supplementation of these cytokines in vitro has the potential to alleviate some of the challenges associated with the cryo-survival of in vitro produced bovine embryos through improving embryo development and embryo quality.
Asunto(s)
Bovinos/embriología , Criopreservación/veterinaria , Embrión de Mamíferos/embriología , Factor 2 de Crecimiento de Fibroblastos , Factor I del Crecimiento Similar a la Insulina , Factor Inhibidor de Leucemia , Animales , Blastocisto/citología , Blastocisto/efectos de los fármacos , Blastocisto/ultraestructura , Criopreservación/métodos , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/ultraestructura , Femenino , Fertilización In Vitro/métodos , Fertilización In Vitro/veterinaria , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Factor 2 de Crecimiento de Fibroblastos/farmacología , Técnicas de Maduración In Vitro de los Oocitos/métodos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor Inhibidor de Leucemia/administración & dosificación , Factor Inhibidor de Leucemia/farmacología , EmbarazoRESUMEN
The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1-/-) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1-/- embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1-/- embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1-/- conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1-/- embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1-/- gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1-/- embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .
Asunto(s)
Embrión de Mamíferos/metabolismo , Estrógenos/metabolismo , Mantenimiento del Embarazo/fisiología , Preñez , Reconocimiento en Psicología/fisiología , Porcinos , Animales , Animales Modificados Genéticamente , Aromatasa/genética , Aromatasa/metabolismo , Células Cultivadas , Clonación de Organismos/veterinaria , Técnicas de Cultivo de Embriones/veterinaria , Transferencia de Embrión/veterinaria , Embrión de Mamíferos/química , Desarrollo Embrionario/efectos de los fármacos , Estrógenos/farmacología , Femenino , Fertilización/fisiología , Intercambio Materno-Fetal/efectos de los fármacos , Intercambio Materno-Fetal/fisiología , Técnicas de Transferencia Nuclear , Embarazo , Mantenimiento del Embarazo/efectos de los fármacos , Reconocimiento en Psicología/efectos de los fármacos , Porcinos/embriología , Porcinos/genética , Porcinos/metabolismoRESUMEN
Genetically engineered pigs serve as excellent biomedical and agricultural models. To date, the most reliable way to generate genetically engineered pigs is via somatic cell nuclear transfer (SCNT), however, the efficiency of cloning in pigs is low (1-3%). Somatic cells such as fibroblasts frequently used in nuclear transfer utilize the tricarboxylic acid cycle and mitochondrial oxidative phosphorylation for efficient energy production. The metabolism of somatic cells contrasts with cells within the early embryo, which predominately use glycolysis. We hypothesized that fibroblast cells could become blastomere-like if mitochondrial oxidative phosphorylation was inhibited by hypoxia and that this would result in improved in vitro embryonic development after SCNT. In a previous study, we demonstrated that fibroblasts cultured under hypoxic conditions had changes in gene expression consistent with increased glycolytic/gluconeogenic metabolism. The goal of this pilot study was to determine if subsequent in vitro embryo development is impacted by cloning porcine embryonic fibroblasts cultured in hypoxia. Here we demonstrate that in vitro measures such as early cleavage, blastocyst development, and blastocyst cell number are improved (4.4%, 5.5%, and 17.6 cells, respectively) when donor cells are cultured in hypoxia before nuclear transfer. Survival probability was increased in clones from hypoxic cultured donors compared to controls (8.5 vs. 4.0 ± 0.2). These results suggest that the clones from donor cells cultured in hypoxia are more developmentally competent and this may be due to improved nuclear reprogramming during somatic cell nuclear transfer.
Asunto(s)
Blastocisto/citología , Técnicas de Cultivo de Célula/métodos , Hipoxia de la Célula/fisiología , Fibroblastos/citología , Técnicas de Transferencia Nuclear , Animales , Blastocisto/fisiología , Células Cultivadas , Reprogramación Celular/fisiología , Clonación de Organismos , Embrión de Mamíferos/citología , Desarrollo Embrionario/fisiología , Femenino , Fibroblastos/fisiología , Proyectos Piloto , Embarazo , PorcinosRESUMEN
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.
Asunto(s)
Clonación de Organismos , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Técnicas de Transferencia Nuclear , Animales , Femenino , Embarazo , PorcinosRESUMEN
Conceptus expansion throughout the uterus of mammalian species with a noninvasive epitheliochorial type of placentation is critical establishing an adequate uterine surface area for nutrient support during gestation. Pig conceptuses undergo a unique rapid morphological transformation to elongate into filamentous threads within 1 h, which provides the uterine surface to support development and maintain functional corpora lutea through the production of estrogen. Conceptus production of a unique interleukin 1ß, IL1B2, temporally increases during the period of trophoblast remodeling during elongation. CRISPR/Cas9 gene editing was used to knock out pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. Trophoblast elongation occurred on day 14 in wild-type (IL1B2+/+) conceptuses but did not occur in ILB2-null (IL1B2-/-) conceptuses. Although the morphological transition of IL1B2-/- conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. However, conceptus aromatase expression and estrogen secretion were decreased, indicating that IL1B2 may be involved in the spatiotemporal increase in conceptus estrogen synthesis needed for the establishment of pregnancy in the pig and may serve to regulate the proinflammatory response of endometrium to IL1B2 during conceptus elongation and attachment to the uterine surface.
Asunto(s)
Proliferación Celular/genética , Interleucina-1beta/genética , Trofoblastos/metabolismo , Útero/metabolismo , Animales , Sistemas CRISPR-Cas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Endometrio/metabolismo , Estrógenos/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Interleucina-1beta/metabolismo , Embarazo , Porcinos , Factores de Tiempo , Trofoblastos/citologíaRESUMEN
Large-scale genomic studies have made major progress in identifying genetic risk variants for schizophrenia. A key finding from these studies is that there is an increased burden of genomic copy number variants (CNVs) in schizophrenia cases compared with controls. The mechanism through which these CNVs confer risk for the symptoms of schizophrenia, however, remains unclear. One possibility is that schizophrenia risk CNVs impact basic associative learning processes, abnormalities of which have long been associated with the disorder. To investigate whether genes in schizophrenia CNVs impact on specific phases of associative learning we combined human genetics with experimental gene expression studies in animals. In a sample of 11 917 schizophrenia cases and 16 416 controls, we investigated whether CNVs from patients with schizophrenia are enriched for genes expressed during the consolidation, retrieval or extinction of associative memories. We show that CNVs from cases are enriched for genes expressed during fear extinction in the hippocampus, but not genes expressed following consolidation or retrieval. These results suggest that CNVs act to impair inhibitory learning in schizophrenia, potentially contributing to the development of core symptoms of the disorder.
Asunto(s)
Aprendizaje por Asociación/fisiología , Variaciones en el Número de Copia de ADN/genética , Esquizofrenia/genética , Animales , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/fisiología , Estudios de Casos y Controles , Condicionamiento Clásico , Bases de Datos Factuales , Miedo/fisiología , Miedo/psicología , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Masculino , RatasRESUMEN
The CRISPR/Cas9 genome editing tool has increased the efficiency of creating genetically modified pigs for use as biomedical or agricultural models. The objectives were to determine if DNA editing resulted in a delay in development to the blastocyst stage or in a skewing of the sex ratio. Six DNA templates (gBlocks) that were designed to express guide RNAs that target the transmembrane protease, serine S1, member 2 (TMPRSS2) gene were in vitro transcribed. Pairs of CRISPR guide RNAs that flanked the start codon and polyadenylated Cas9 were co-injected into the cytoplasm of zygotes and cultured in vitro to the blastocyst stage. Blastocysts were collected as they formed on days 5, 6 or 7. PCR was performed to determine genotype and sex of each embryo. Separately, embryos were surgically transferred into recipient gilts on day 4 of estrus. The rate of blastocyst development was not significantly different between CRISPR injection embryos or the non-injected controls at day 5, 6 or 7 (p = 0.36, 0.09, 0.63, respectively). Injection of three CRISPR sets of guides resulted in a detectable INDEL in 92-100 % of the embryos analyzed. There was not a difference in the number of edits or sex ratio of male to female embryos when compared between days 5, 6 and 7 to the controls (p > 0.22, >0.85). There were 12 resulting piglets and all 12 had biallelic edits of TMRPSS2. Zygote injection with CRISPR/Cas9 continues to be a highly efficient tool to genetically modify pig embryos.
Asunto(s)
Desarrollo Embrionario/genética , Marcación de Gen/métodos , Porcinos/genética , Cigoto/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/crecimiento & desarrollo , Blastocisto/metabolismo , Sistemas CRISPR-Cas/genética , ARN Guía de Kinetoplastida/genética , Razón de Masculinidad , Porcinos/crecimiento & desarrolloRESUMEN
In this study, we described the phenotype of monoallelic interleukin 2 receptor gamma knockout (mIL2RG+/Δ69-368 KO) pigs. Approximately 80% of mIL2RG+/Δ69-368 KO pigs (8/10) were athymic, whereas 20% (2/10) presented a rudimentary thymus. The body weight of IL2RG+/Δ69-368KO pigs developed normally. Immunological analysis showed that mIL2RG+/Δ69-368 KO pigs possessed CD25+CD44- or CD25-CD44+ cells, whereas single (CD4 or CD8) or double (CD4/8) positive cells were lacking in mIL2RG+/Δ69-368 KO pigs. CD3+ cells in the thymus of mIL2RG+/Δ69-368 KO pigs contained mainly CD44+ cells and/or CD25+ cells, which included FOXP3+ cells. These observations demonstrated that T cells from mIL2RG+/Δ69-368 KO pigs were able to develop to the DN3 stage, but failed to transition toward the DN4 stage. Whole-transcriptome analysis of thymus and spleen, and subsequent pathway analysis revealed that a subset of genes differentially expressed following the loss of IL2RG might be responsible for both impaired T-cell receptor and cytokine-mediated signalling. However, comparative analysis of two mIL2RG+/Δ69-368 KO pigs revealed little variability in the down- and up-regulated gene sets. In conclusion, mIL2RG+/Δ69-368 KO pigs presented a T-B+NK- SCID phenotype, suggesting that pigs can be used as a valuable and suitable biomedical model for human SCID research.
Asunto(s)
Modelos Animales de Enfermedad , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Inmunodeficiencia Combinada Grave , Animales , Técnicas de Inactivación de Genes , Humanos , Subunidad gamma Común de Receptores de Interleucina/inmunología , PorcinosRESUMEN
Cryostorage of porcine embryos in a closed pathogen-free system is essential for the maintenance and safeguard of swine models. Previously, we reported a protocol for the successful cryopreservation of porcine embryos at the blastocyst stage in 0.25 mL ministraws. In this experiment, we aimed at developing a protocol to apply the same concept for the cryopreservation of early-stage porcine embryos. Porcine embryos from day 2 through day 4 were delipidated by using a modified two-step centrifugation method and were then cryopreserved in sealed 0.25 mL straws by using a slow cooling method. Control groups included open pulled straw (OPS) vitrified embryos after delipidation and noncryopreserved embryos without delipidation. There were no significant differences in cryosurvival between embryos frozen in 0.25 mL straws and OPS vitrified embryos across all the stages (two cell to morula) examined (p>0.05). Similarly, in all groups examined, the blastocyst rates were not different between the two cryopreserved groups. However, the blastocyst rates from the cryopreserved groups were significantly lower than the noncryopreserved controls (p<0.05). This experiment demonstrated that early-stage porcine embryos can survive cryopreservation in a closed system by using a slow cooling method at a comparable rate to those vitrified by using an ultrarapid cooling method (p>0.05). However, the developmental competence was significantly reduced after cryopreservation compared to noncryopreserved embryos. Further research is needed to optimize the protocol to improve the developmental potential of cryopreserved early-stage porcine embryos in sealed straws.
RESUMEN
Artificial oocyte activation is a critical step during SCNT. Most current activation protocols focus on inducing an increase in the intracellular free Ca(2+) concentration of the oocyte. Here, we have used a zinc chelator, TPEN, to enhance the efficiency of oocyte activation during SCNT. TPEN treatment of matured pig oocytes resulted in the reduction of available Zn(2+) in pig oocytes; however, the cytosolic Ca(2+) concentration in the oocytes was not affected by the TPEN treatment. When various concentrations (100-250 µM) and incubation durations (45 minutes-2.5 hours) of TPEN were used to activate oocytes, the efficiency of oocyte activation was not different from conventional activation methods. When oocytes that were activated by conventional activation methods were incubated with a lower concentration of TPEN (5-10 µM), a significant increase in embryos developing to the blastocyst stage was observed. In addition, when oocytes receiving a small Ca(2+) stimulus were further activated by higher concentration of TPEN (100-200 µM), a significant increase in the frequency of blastocyst formation was observed, compared to a conventional activation method. This result indicated that TPEN can be a main reagent in oocyte activation. No increase in the cytosolic Ca(2+) level was detected when oocytes were exposed to various concentrations of TPEN, indicating the ability of TPEN to induce oocyte activation is independent of an intracellular Ca(2+) increase. We were able to produce clones through SCNT by using the TPEN-assisted activation procedure, and the piglets produced through the process did not show any signs of abnormality. In this study, we have developed an efficient way to use TPEN to increase the developmental potential of cloned embryos.
Asunto(s)
Etilenodiaminas/farmacología , Técnicas de Transferencia Nuclear/veterinaria , Oocitos/efectos de los fármacos , Porcinos/fisiología , Zinc/química , Animales , Calcio/metabolismo , Desarrollo Embrionario , Técnicas de Maduración In Vitro de los Oocitos/métodos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/metabolismo , Oocitos/fisiologíaRESUMEN
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.
Asunto(s)
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
Faulty epigenetic reprogramming of somatic nuclei is thought to be the main reason for low cloning efficiency by somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi), such as Scriptaid, improve developmental competence of SCNT embryos in several species. Another HDACi, Oxamflatin, is about 100 times more potent than Scriptaid in the ability to inhibit nuclear-specific HDACs. The present study determined the effects of Oxamflatin treatment on embryo development, DNA methylation, and gene expression. Oxamflatin treatment enhanced blastocyst formation of SCNT embryos in vitro. Embryo transfer produced more pigs born and fewer mummies from the Oxamflatin-treated group compared to the Scriptaid-treated positive control. Oxamflatin also decreased DNA methylation of POU5F1 regulatory elements and centromeric repeat elements in day-7 blastocysts. When compared to in vitro-fertilized (IVF) embryos, the methylation status of POU5F1, NANOG, and centromeric repeat was similar in the cloned embryos, indicating these genes were successfully reprogrammed. However, compared to the lack of methylation of XIST in day-7 IVF embryos, a higher methylation level in day-7 cloned embryos was observed, implying that X chromosomes were activated in day-7 IVF blastocysts, but were not fully activated in cloned embryos, i.e., reprogramming of XIST was delayed. A time-course analysis of XIST DNA methylation on day-13, -15, -17, and -19 in vivo embryos revealed that XIST methylation initiated at about day 13 and was not completed by day 19. The methylation of the XIST gene in day-19 control cloned embryos was delayed again when compared to in vivo embryos. However, methylation of XIST in Oxamflatin-treated embryos was comparable with in vivo embryos, which further demonstrated that Oxamflatin could accelerate the delayed reprogramming of XIST gene and thus might improve cloning efficiency.
Asunto(s)
Blastocisto/citología , Reprogramación Celular/efectos de los fármacos , Clonación de Organismos/métodos , Desarrollo Embrionario/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Animales , Metilación de ADN , Técnicas de Cultivo de Embriones , Transferencia de Embrión/veterinaria , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Proteínas de Homeodominio/metabolismo , Hidroxilaminas/farmacología , Masculino , Técnicas de Transferencia Nuclear/veterinaria , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Embarazo , Quinolinas/farmacología , ARN Largo no Codificante/metabolismo , PorcinosRESUMEN
Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.
Asunto(s)
Animales Modificados Genéticamente/fisiología , Blastocisto/fisiología , Sistemas CRISPR-Cas , Embrión de Mamíferos/fisiología , Ingeniería Genética/veterinaria , Oocitos/fisiología , Sus scrofa/fisiología , Animales , Animales Modificados Genéticamente/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD1d/química , Antígenos CD1d/genética , Antígenos CD1d/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Línea Celular , Técnicas de Cultivo de Embriones/veterinaria , Transferencia de Embrión/veterinaria , Femenino , Fertilización In Vitro/veterinaria , Eliminación de Gen , Ingeniería Genética/efectos adversos , Ingeniería Genética/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Masculino , Mutación , Técnicas de Transferencia Nuclear/veterinaria , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Sus scrofa/genética , TransgenesRESUMEN
Pigs with severe combined immunodeficiency (SCID) may provide useful models for regenerative medicine, xenotransplantation, and tumor development and will aid in developing therapies for human SCID patients. Using a reporter-guided transcription activator-like effector nuclease (TALEN) system, we generated targeted modifications of recombination activating gene (RAG) 2 in somatic cells at high efficiency, including some that affected both alleles. Somatic-cell nuclear transfer performed with the mutated cells produced pigs with RAG2 mutations without integrated exogenous DNA. Biallelically modified pigs either lacked a thymus or had one that was underdeveloped. Their splenic white pulp lacked B and T cells. Under a conventional housing environment, the biallelic RAG2 mutants manifested a "failure to thrive" phenotype, with signs of inflammation and apoptosis in the spleen compared with age-matched wild-type animals by the time they were 4 wk of age. Pigs raised in a clean environment were healthier and, following injection of human induced pluripotent stem cells (iPSCs), quickly developed mature teratomas representing all three germ layers. The pigs also tolerated grafts of allogeneic porcine trophoblast stem cells. These SCID pigs should have a variety of uses in transplantation biology.
Asunto(s)
Proteínas de Unión al ADN/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Proteínas Nucleares/genética , Inmunodeficiencia Combinada Grave/metabolismo , Trasplante Heterólogo , Alelos , Animales , Secuencia de Bases , Fibroblastos/metabolismo , Genotipo , Humanos , Datos de Secuencia Molecular , Mutación , Fenotipo , Regeneración , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/terapia , Porcinos , Porcinos Enanos , Timo/metabolismo , Cordón Umbilical/citologíaRESUMEN
The ability to mature oocytes in vitro provides a tool for creating embryos by parthenogenesis, fertilization, and cloning. Unfortunately the quality of oocytes matured in vitro falls behind that of in vivo matured oocytes. To address this difference, transcriptional profiling by deep sequencing was conducted on pig oocytes that were either matured in vitro or in vivo. Alignment of over 18 million reads identified 1,316 transcripts that were differentially represented. One pathway that was overrepresented in the oocytes matured in vitro was for Wingless-type MMTV integration site (WNT) signaling. In an attempt to inhibit the WNT pathway, Dickkopf-related protein 1 was added to the in vitro maturation medium. Addition of Dickkopf-related protein 1 improved the percentage of oocytes that matured to the metaphase II stage, increased the number of nuclei in the resulting blastocyst stage embryos, and reduced the amount of disheveled segment polarity protein 1 protein in oocytes. It is concluded that transcriptional profiling is a powerful method for detecting differences between in vitro and in vivo matured oocytes, and that the WNT signaling pathway is important for proper oocyte maturation.
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Péptidos y Proteínas de Señalización Intercelular/genética , Oocitos/metabolismo , Oogénesis/genética , Partenogénesis/genética , Proteínas Wnt/genética , Vía de Señalización Wnt/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Blastocisto/citología , Blastocisto/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Dishevelled , Transferencia de Embrión , Femenino , Fertilización In Vitro , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Metafase , Oocitos/citología , Oocitos/crecimiento & desarrollo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Porcinos , Transcriptoma , Proteínas Wnt/metabolismoRESUMEN
In vitro embryo production is important for research in animal reproduction, embryo transfer, transgenics, and cloning. Yet, in vitro-fertilized (IVF) embryos are generally developmentally delayed and are inferior to in vivo-derived (IVV) embryos; this discrepancy is likely a result of aberrant gene expression. Transcription of three genes implicated to be important in normal preimplantation embryo development, TRIM28, SETDB1, and TP53, was determined by quanitative PCR in IVF, somatic-cell nuclear transfer (SCNT), parthenogenetic, and IVV porcine oocytes and embryos. There was no difference in TRIM28 or SETDB1 abundance between oocytes matured in vitro versus in vivo (P > 0.05), whereas TP53 levels were higher in in vitro-matured oocytes. TRIM28 increased from metaphase-II oocytes to the 4-cell and blastocyst stages in IVF embryos, whereas IVV embryos showed a reduction in TRIM28 abundance from maturation throughout development. The relative abundance of TP53 increased by the blastocyst stage in all treatment groups, but was higher in IVF embryos compared to IVV and SCNT embryos. In contrast, SETDB1 transcript levels decreased from the 2-cell to blastocyst stage in all treatments. For each gene analyzed, SCNT embryos of both hard-to-clone and easy-to-clone cell lines were more comparable to IVV than IVF embryos. Knockdown of TRIM28 also had no effect on blastocyst development or expression of SETDB1 or TP53. Thus, TRIM28, SETDB1, and TP53 are dynamically expressed in porcine oocytes and embryos. Furthermore, TRIM28 and TP53 abundances in IVV and SCNT embryos are similar, but different from quantities in IVF embryos.
Asunto(s)
Blastocisto/metabolismo , Clonación de Organismos , Fertilización In Vitro , Regulación del Desarrollo de la Expresión Génica , Técnicas de Transferencia Nuclear , Proteína Metiltransferasas/biosíntesis , Proteínas Represoras/biosíntesis , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Blastocisto/patología , Femenino , Partenogénesis , PorcinosRESUMEN
The possibility of fertilization without male contribution to the embryonic genome was investigated in pig oocytes. Mature oocytes were co-incubated with sperm, and in an attempt to prevent the incorporation of the sperm head into the ooplasm, the actin polymerization inhibitor cytochalasin B was added to the fertilization medium. We found that perturbing actin filament integrity did not affect the pattern of the sperm-induced Ca(2+) signal or the process of cortical granule exocytosis, and it did not alter the percentage of activated oocytes compared to the control (oocytes fertilized in the absence of the inhibitor). However, over 20% of the cytochalasin B-treated oocytes formed only a single pronucleus after fertilization, indicating that the inhibitor blocked sperm head incorporation at least in some oocytes. In most cases, cytochalasin B also prevented the integration of the male chromosomes into the embryonic genome as determined by the absence of the SRY gene in the embryonic blastomeres or by the frequency of embryos showing green fluorescence after sperm from a GFP-transgenic boar was used for fertilization. Finally, the percentage of embryos that developed beyond the four-cell stage and the total number of nuclei in the resultant blastocysts were higher when oocytes reconstructed by nuclear transfer were activated by fertilization in the presence of cytochalasin B compared to the control group, where activation was induced by electroporation. These results suggest that fertilization in the presence of cytochalasin B can induce oocyte activation while it also prevents integration of the male genome into the embryo. This method has the potential to be used as an alternative to inducing embryonic development after nuclear transfer.
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Blastómeros/metabolismo , Citocalasina B/farmacología , Desarrollo Embrionario/efectos de los fármacos , Fertilización In Vitro , Oocitos/metabolismo , Animales , Blastómeros/citología , Calcio/metabolismo , Exocitosis/efectos de los fármacos , Femenino , Masculino , Oocitos/citología , Espermatozoides/citología , Espermatozoides/metabolismo , PorcinosRESUMEN
Dynamic changes in DNA methylation are observed during embryo development. Recent studies show that the TET family is involved in these changes by converting 5-methylcytosine (5mec) to 5-hydroxymethylcytosine (5hmec). Specifically, TET3 is responsible for the conversion in the early stages, and then TET1 is a key regulator at later stages of embryo development. From previous mouse reports and our preliminary data in porcine embryos, we hypothesized that TET1 becomes the main regulator at the time of the maternal to zygotic transition (MZT). Transcript abundance of TET3 was high only at the zygote and 2-cell stage. The abundance of TET1 mRNA was high in the blastocysts and TET1 protein was present at the 4-cell stage and the blastocysts. The dynamic was similar in porcine somatic cell nuclear transfer (SCNT) embryos however; abnormally upregulated TET3 was detected at the 4-cell stage. When transcription or translation was blocked at the 2-cell stage, TET3 mRNA remained high at the 4-cell stage suggesting that degradation of TET3 is related to the MZT. Downregulation of TET3 before fertilization resulted in the reduction of 5hmec in zygotes indicating that TET3 is a key molecule for 5hmec synthesis. This misregulation of 5hmec in zygotes also affected the level of NANOG expression in the blastocysts. We show here that the porcine TET family shows dynamic expression patterns during embryogenesis, and is responsible for the appearance of 5hmec in the zygotes by TET3. This appearance of 5hmec in zygote is important for the expression of NANOG in the blastocysts.
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Blastocisto/metabolismo , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Proto-Oncogénicas/genética , Animales , Dioxigenasas/genética , Fertilización , Fertilización In Vitro , Humanos , Inmunohistoquímica , Ratones , Oxigenasas de Función Mixta , Familia de Multigenes , Proteína Homeótica Nanog , Técnicas de Transferencia Nuclear , Oocitos/citología , ARN Mensajero/metabolismo , Especificidad de la Especie , Porcinos , Cigoto/metabolismoRESUMEN
Surface expression of SIGLEC1, also known as sialoadhesin or CD169, is considered a primary determinant of the permissiveness of porcine alveolar macrophages for infection by porcine reproductive and respiratory syndrome virus (PRRSV). In vitro, the attachment and internalization of PRRSV are dependent on the interaction between sialic acid on the virion surface and the sialic acid binding domain of the SIGLEC1 gene. To test the role of SIGLEC1 in PRRSV infection, a SIGLEC1 gene knockout pig was created by removing part of exon 1 and all of exons 2 and 3 of the SIGLEC1 gene. The resulting knockout ablated SIGLEC1 expression on the surface of alveolar macrophages but had no effect on the expression of CD163, a coreceptor for PRRSV. After infection, PRRSV viremia in SIGLEC1(-/-) pigs followed the same course as in SIGLEC1(-/+) and SIGLEC1(+/+) littermates. The absence of SIGLEC1 had no measurable effect on other aspects of PRRSV infection, including clinical disease course and histopathology. The results demonstrate that the expression of the SIGLEC1 gene is not required for infection of pigs with PRRSV and that the absence of SIGLEC1 does not contribute to the pathogenesis of acute disease.