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A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.
Assuntos
Macrófagos Alveolares , Células-Tronco Pluripotentes , Suínos , Animais , Endocitose , Hematopoese/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Mesoderma/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Transdução de Sinais/efeitos dos fármacos , Suínos/virologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fatores de TempoRESUMO
As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.
Assuntos
Células-Tronco Pluripotentes Induzidas , Vírus da Síndrome Respiratória e Reprodutiva Suína , Suínos , Animais , Células-Tronco Pluripotentes Induzidas/metabolismo , Receptores de Superfície Celular/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/genéticaRESUMO
Background: With the growth of trajectory data, the large amount of data causes a lot of problems with storage, analysis, mining, etc. Most of the traditional trajectory data compression methods are focused on preserving spatial characteristic information and pay little attention to other temporal information on trajectory data, such as speed change points or stop points. Methods: A data compression algorithm based on the spatio-temporal characteristics (CASC) of the trajectory data is proposed to solve this problem. This algorithm compresses trajectory data by taking the azimuth difference, velocity difference and time interval as parameters in order to preserve spatial-temporal characteristics. Microsoft's Geolife1.3 data set was used for a compression test to verify the validity of the algorithm. The compression results were compared with the traditional Douglas-Peucker (DP), Top-Down Time Ratio (TD-TR) and Opening Window (OPW) algorithms. Compression rate, the direction information of trajectory points, vertical synchronization distance, and algorithm type (online/offline) were used to evaluate the above algorithms. Results: The experimental results show that with the same compression rate, the ability of the CASC to retain the forward direction trajectory is optimal, followed by TD-TR, DP, and then OPW. The velocity characteristics of the trajectories are also stably retained when the speed threshold value is not more than 100%. Unlike the DP and TD-TR algorithms, CASC is an online algorithm. Compared with OPW, which is also an online algorithm, CASC has better compression quality. The error distributions of the four algorithms have been compared, and CASC is the most stable algorithm. Taken together, CASC outperforms DP, TD-TR and OPW in trajectory compression.
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Porcine embryonic fibroblasts (PEFs) can be directly reprogrammed into porcine induced pluripotent stem cells (piPSCs). However, the reprogramming process is generally lengthy and inefficient. Here, we established a fast and efficient induction system of piPSCs from porcine Sertoli cells (SCs) via forced expression of pig Yamanaka factors. The alkaline phosphatase (AP)-positive colonies from SCs developed on Day 3 after lentivirus infection, and were expanded and then picked up on Day 7, whereas reprogramming process from PEFs did not show any colonies in the same period. The picked piPSCs strongly expressed pluripotent genes, had the differentiation capacity to three germ layers, and could be also induced into primordial germ cell-like cells. Screening for transcription factor combinations showed that POU class 5 homeobox 1 (OCT4) is the core factor for AP-positive colony formation, and two factors (OCT4 and c-MYC) could successfully reprogram SCs into piPSCs. We then compared the RNA-sequencing data of piPSCs derived from SCs and PEFs, and found that the most significant difference was the activation of Transforming Growth Factor ß signaling pathway. We also compared the RNA levels of SCs and PEFs, and found that SCs exhibited higher Wnt signaling activity and Bone Morphogenetic Protein 4 expression than PEFs, which might be correlated with higher cell proliferation rate and reprogramming efficiency. In summary, the data demonstrated that starting cell sources of piPSCs significantly affect reprogramming dynamics and SCs could serve as cell sources for efficient reprogramming.
Assuntos
Reprogramação Celular , Fibroblastos , Células-Tronco Pluripotentes Induzidas , Células de Sertoli , Animais , Masculino , Diferenciação Celular , Células Cultivadas , Fibroblastos/citologia , Células-Tronco Pluripotentes Induzidas/citologia , RNA/genética , Células de Sertoli/citologia , SuínosRESUMO
Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share an evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that displayed remarkable conservation in placental mammals and were sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR-Cas9 approach severely impaired stem cell pluripotency. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals.
Assuntos
Elementos Facilitadores Genéticos , Células-Tronco Pluripotentes , Animais , Proteínas de Ciclo Celular/metabolismo , Elementos Facilitadores Genéticos/genética , Eutérios/genética , Feminino , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Placenta/metabolismo , Células-Tronco Pluripotentes/metabolismo , Gravidez , Suínos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Studies on ESRRB-regulating porcine-induced pluripotent stem cells (piPSCs) converted to trophoblast-like stem cells (TLSCs) contribute to the understanding of early embryo development. However, the epigenetic modification regulation network during the conversion is poorly understood. Here, the global change in histone H3 Lysine 4, 9, 27, 36 methylation and Lysine 27 acetylation was investigated in piPSCs and TLSCs. We found a high modification profile of H3K36me2 in TLSCs compared to that of piPSCs, whereas the profiles of other modifications remained constant. KDM4C, a H3K36me3/2 demethylase, whose gene body region was combined with ESRRB, was upregulated in TLSCs. Moreover, KDM4 inhibitor supplementation rescued the AP-negative phenotype observed in TLSCs, confirming that KDM4C could regulate the pluripotency of TLSCs. Subsequently, KDM4C replenishment results show the significantly repressed proliferation and AP-positive staining of TLSCs. The expressions of CDX2 and KRT8 were also upregulated after KDM4C overexpression. In summary, these results show that KDM4C replaced the function of ESRRB. These findings reveal the unique and crucial role of KDM4C-mediated epigenetic chromatin modifications in determination of piPSCs' fate and expand the understanding of the connection between piPSCs and TSCs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Animais , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisina/metabolismo , Metilação , Células-Tronco Pluripotentes/metabolismo , Suínos , Trofoblastos/metabolismoRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro passaging leads to stemness loss of MSCs, resulting in failure of MSC therapy. This study investigated whether the combination of melatonin and human umbilical cord mesenchymal stem cells (hUC-MSCs) was superior to hUC-MSCs alone in ameliorating high-fat diet and streptozocin (STZ)-induced type II diabetes mellitus (T2DM) in a mouse model. METHODS: Mice were divided into four groups: normal control (NC) group; T2DM group; hUC-MSCs treatment alone (UCMSC) group and pretreatment of hUC-MSCs with melatonin (UCMSC/Mel) group. RESULTS: RNA sequence analysis showed that certain pathways, including the signaling pathway involved in the regulation of cell proliferation signaling pathway, were regulated by melatonin. The blood glucose levels of the mice in the UCMSC and UCMSC/Mel treatment groups were significantly reduced compared with the T2DM group without treatment (P < 0.05). Furthermore, hUC-MSCs enhance the key factor in the activation of the PI3K/Akt pathway in T2DM mouse hepatocytes. CONCLUSION: The pretreatment of hUC-MSCs with melatonin partly boosted cell efficiency and thereby alleviated impaired glycemic control and insulin resistance. This study provides a practical strategy to improve the application of hUC-MSCs in diabetes mellitus and cytotherapy. Overview of the PI3K/AKT signaling pathway. (A) Underlying mechanism of UCMSC/Mel inhibition of hyperglycemia and insulin resistance T2DM mice via regulation of PI3K/AKT pathway. hUC-MSCs stimulates glucose uptake and improves insulin action thus should inhibition the clinical signs of T2DM, through activation of the p-PI3K/Akt signaling pathway and then regulates glucose transport through activating AS160. UCMSC/Mel increases p53-dependent expression of BCL2, and inhibit BAX and Capase3 protein activation. Leading to the decrease in apoptosis. (B) Melatonin modulated PI3K/AKT signaling pathway. Melatonin activated PI3K/AKT response pathway through binding to MT1and MT2 receptor. Leading to the increase in hUC-MSCs proliferation, migration and differentiation. â (Direct stimulatory modification); â´ ( Direct Inhibitory modification); â ⤠(Multistep inhibitory modification); ↑ (Up regulate); ↓ (Down regulate); PI3K (Phosphoinositide 3-Kinase); AKT ( protein kinase B); PDK1 (Phosphoinositide-dependent protein kinase 1); IR, insulin receptor; GLUT4 ( glucose transporter type 4); ROS (reactive oxygen species); BCL-2 (B-cell lymphoma-2); PDK1 (phosphoinositide-dependent kinase 1) BAX (B-cell lymphoma-2-associated X protein); PCNA (Proliferating cell nuclear antigen); Cell cycle-associated proteins (KI67, cyclin A, cyclin E).
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Melatonina , Transplante de Células-Tronco Mesenquimais , Animais , Diabetes Mellitus Tipo 2/terapia , Humanos , Melatonina/farmacologia , Melatonina/uso terapêutico , Transplante de Células-Tronco Mesenquimais/métodos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/uso terapêutico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Cordão Umbilical , Proteína X Associada a bcl-2RESUMO
The establishment of porcine pluripotent stem cells (piPSCs) is critical but remains challenging. All piPSCs are extremely sensitive to minor perturbations of culture conditions and signaling network. Inhibitors, such as CHIR99021 and XAV939 targeting the WNT signaling pathway, have been added in a culture medium to modify the cell regulatory network. However, potential side effects of inhibitors could confine the pluripotency and practicability of piPSCs. This study aimed to investigate the roles of AXIN, one component of the WNT pathway in piPSCs. Here, porcine AXIN1 and AXIN2 genes were knocked-down or overexpressed. Digital RNA-seq was performed to explore the mechanism of cell proliferation and apoptosis. We found that (1) overexpression of the porcine AXIN2 gene significantly reduced survival and negatively impacted the pluripotency of piPSCs, and (2) knockdown of AXIN2, a negative effector of the WNT signaling pathway, enhanced the expression of genes involved in cell cycle but reduced the expression of genes related to cell differentiation, death, and apoptosis.
Assuntos
Proteína Axina/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Proteína Axina/deficiência , Proteína Axina/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proliferação de Células/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Técnicas de Silenciamento de Genes , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Modelos Biológicos , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq , Suínos , Regulação para Cima , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Porcine-induced pluripotent stem cells (piPSCs) could serve as a great model system for human stem cell preclinical research. However, the pluripotency gene network of piPSCs, especially the function for the core transcription factor estrogen-related receptor beta (ESRRB), was poorly understood. Here, we constructed ESRRB-overexpressing piPSCs (ESRRB-piPSCs). Compared with the control piPSCs (CON-piPSCs), the ESRRB-piPSCs showed flat, monolayered colony morphology. Moreover, the ESRRB-piPSCs showed greater chimeric capacity into trophectoderm than CON-piPSCs. We found that ESRRB could directly regulate the expressions of trophoblast stem cell (TSC)-specific markers, including KRT8, KRT18 and CDX2, through binding to their promoter regions. Mutational analysis proved that the N-terminus zinc finger domain is indispensable for ESRRB to regulate the TSC markers. Furthermore, this regulation needs the participation of OCT4. Accordingly, the cooperation between ESRRB and OCT4 facilitates the conversion from pluripotent state to the trophoblast-like state. Our results demonstrated a unique and crucial role of ESRRB in determining piPSCs fate, and shed new light on the molecular mechanism underlying the segregation of embryonic and extra-embryonic lineages.
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MicroRNAs are important regulators in stem cells, which involve in gene regulation, including cell proliferation, differentiation and apoptosis. As an important one, miR-34c participates in various processes by targeting protein-coding genes. It is generally considered as a tumor suppressor and cell adhesion inhibitor. However, whether miR-34c has effects on pluripotent stem cells is not clear. Here, by mir-34c mimics transfection, the function of miR-34c on porcine induced pluripotent stem cell (piPSC)-like cells was investigated. Bioinformatics analyses showed that c-Myc is miR-34c's candidate target, which was confirmed by dual Luciferase assay. The knockout of miR-34c indicated that mir-34c affects the proliferation and pluripotency of piPSC-like cells by targeting c-Myc. Our study explored the regulatory mechanism of miR-34c on piPSC-like cells, providing a reference for the establishment of true porcine PSCs.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Proliferação de Células/genética , Regulação da Expressão Gênica , Camundongos , MicroRNAs/genética , SuínosRESUMO
The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs.
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Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Células-Tronco Pluripotentes Induzidas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Metilação de DNA , Epigênese Genética , Células-Tronco Pluripotentes Induzidas/citologia , Histona Desmetilases com o Domínio Jumonji/genética , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , SuínosRESUMO
LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear. Here, we explored the function of LIN28A in piPSCs based on its overexpression and knockdown. We performed total RNA sequencing (RNA-seq) of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence staining. Results indicated that piPSC proliferation ability decreased following LIN28A knockdown. Furthermore, when LIN28A expression in the shLIN28A2 group was lower (by 20%) than that in the negative control knockdown group ( shNC), the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells. Results also showed that LIN28A overexpression inhibited the expression of DUSP (dual-specificity phosphatases) family phosphatases and activated the mitogen-activated protein kinase (MAPK) signaling pathway. Thus, LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our study provides a new resource for exploring the functions of LIN28A in piPSCs.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proliferação de Células , Fosfatases de Especificidade Dupla/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas de Ligação a RNA/genética , SuínosRESUMO
OBJECTIVES: The establishment of porcine pluripotent stem cells (pPSCs) is still a critical topic. However, all pPSCs were failed to contribute to efficient chimeric pig and were extremely sensitive to changes of culture conditions. This study aimed to investigate the role of BCL2 in pPSCs and further explain the mechanism. MATERIALS AND METHODS: Porcine BCL2 gene was cloned and overexpressed in porcine induce pluripotent stem cells (piPSCs). Digital RNA-seq was performed to explain the mechanism of anti-apoptosis. Finally, the cells carrying BCL2 were injected into mouse early embryo to evaluate its chimeric ability. RESULTS: Here, we found that overexpression of porcine BCL2 gene significantly improved the survivability of piPSCs and the efficiency of embryonic chimerism, and did not wreck the pluripotency of piPSCs. Furthermore, the Digital RNA-seq analysis revealed that BCL2, as a downstream gene of the PI3K signal pathway, enhanced the expression of PI3K signal pathway receptors, such as FGFR2, and further promoted oxidoreductases activity and lipid metabolism, thus maintaining the survival and pluripotency of piPSCs. CONCLUSION: Our data not only suggested that porcine BCL2 gene could enhance the survivability and chimeric ability of pPSCs, but also explained the positive feedback mechanism in this process, providing strong support for the chimeric experiment of pPSCs.
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Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos , Células-Tronco Pluripotentes/citologia , Gravidez , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , SuínosRESUMO
Complex biochemical reactions take place in the nucleus all the time. Transcription machines must follow the rules. The chromatin state, especially the three-dimensional structure of the genome, plays an important role in gene regulation and expression. The super enhancers are important for defining cell identity in mammalian developmental processes and human diseases. It has been shown that the major components of transcriptional activation complexes are recruited by super enhancer to form phase-separated condensates. We summarize the current knowledge about super enhancer in the 3D genome. Furthermore, a new related transcriptional regulation model from super enhancer is outlined to explain its role in the mammalian cell progress.
Assuntos
Elementos Facilitadores Genéticos/genética , Genoma , Animais , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Metilação de DNA , Humanos , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Since the birth of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, the new genome engineering technology has become a hot topic in the scientific community. However, for swine, the system of pig cells' homology directed repair (HDR) is generally unstable and costly. Here, we aim to make knock-in of porcine cells more realizable. The Rosa26 locus was chosen for gene editing. Through the optimization of strategy, an efficient sgRNA was selected by TIDE analysis. Correspondingly, a vector system was constructed for gene insertion in pRosa26 locus by homologous recombination. A large percentage of cells whose gene is edited easily result in apoptosis. To improve the positive rate, culturing systems have been optimized. Sequence alignment and nuclear transfer confirmed that we got two knock-in cell lines and transgene primary porcine fetal fibroblasts (PFFs) successfully. Results showed that the gene editing platform we used can obtain genetically modified pig cells stably and efficiently. This system can contribute to pig gene research and production of transgenic pigs.
Assuntos
Sistemas CRISPR-Cas , Animais , Linhagem Celular , Edição de Genes , Suínos , TransgenesRESUMO
Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function for continual spermatogenesis. Studies of in vitro culture of germline stem cells are important and meaningful for basic biological research and practical application. Growth factors, such as GDNF, bFGF, CSF1, and EGF, could maintain the self-renewal of mGSCs. Insulin-like growth factor 1 (IGF-1), an important growth factor, and its pathway have been reported to maintain the survival of several types of stem cells and play important roles in male reproduction. However, the mechanism through which the IGF-1 pathway acts to regulate the self-renewal of mGSCs remains unclear. We analyzed the effect of IGF-1 on the proliferation and apoptosis of bovine mGSCs. We evaluated the expression profile of long noncoding RNA (LncRNA) H19 in bovine and mouse tissues. Moreover, we investigated whether LncRNA H19 could regulate the IGF-1 pathway. Results showed that IGF-1 could activate the phosphorylation of AKT and ERK signaling pathways, and the IGF-1 pathway played an important role in regulating the proliferation and apoptosis of bovine mGSCs. The proliferation rate of mGSCs decreased, whereas the apoptosis rate of mGSCs increased when the IGF-1 receptor (IGF-1R) was blocked using the IGF-1R-specific inhibitor (picropodophyllin). LncRNA H19 could regulate the IGF-1 signaling pathway and, consequently, the proliferation and apoptosis of mGSCs. The number of cells in the seminiferous tubule decreased when H19 was interfered by injecting a virus-containing supernatant. Hence, LncRNA H19 participated in the regulation of the proliferation and apoptosis of mGSCs via the IGF-1 signaling pathway.