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PubMed2026

Lipid Metabolism-related lncRNA Model Identifies AC026412.3 as a Driver of Fatty Acid β-oxidation in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Dysregulated lipid metabolism contributes to hepatocellular carcinoma (HCC) progression, but the prognostic value and mechanistic roles of lipid metabolism-related long noncoding RNAs (LRLs) remain insufficiently characterized. This study aimed to construct and validate an LRL-based prognostic model and to investigate the biological function and metabolic mechanism of AC026412.3 in HCC. METHODS: Transcriptomic and clinical data from the The Cancer Genome Atlas Liver Hepatocellular Carcinoma cohort were analyzed to identify LRLs based on their correlation with curated lipid metabolism genes. Differential expression, univariate Cox, least absolute shrinkage and selection operator (LASSO), and multivariate Cox analyses were performed to construct a prognostic signature, which was evaluated using Kaplan-Meier survival and time-dependent receiver operating characteristic (ROC) analyses. Functional enrichment analyses Gene Ontology [GO], Kyoto Encyclopedia of Genes and Genomes [KEGG] and gene set enrichment analysis [GSEA], mutation profiling, tumor mutational burden, immune infiltration estimation, and consensus clustering were applied to characterize associated features. A key LRL was identified through integrated bioinformatic screening and prioritization. Its biological role was assessed by quantitative reverse transcription polymerase chain reactionq (RT-PCR), western blotting, BODIPY staining, colony formation, Transwell assays, and xenograft models. RNA sequencing followed by pathway enrichment analysis was conducted to explore underlying mechanisms. RESULTS: A three-LRL signature (AL031985.3, NRAV, and AC026412.3) stratified HCC patients into distinct risk groups with significantly different survival outcomes and demonstrated independent prognostic value. AC026412.3 was markedly upregulated in HCC and associated with poor prognosis. Functional assays demonstrated that AC026412.3 promoted proliferation, invasion, and tumor growth while reducing lipid accumulation. Mechanistically, AC026412.3 upregulated solute carrier family 22 member 5 (SLC22A5), enhanced fatty acid β-oxidation, and increased adenosine triphosphate (ATP) production, thereby driving metabolic reprogramming. CONCLUSIONS: This study establishes a robust LRL-based prognostic model and identifies AC026412.3 as a key regulator of lipid metabolic reprogramming via the SLC22A5-fatty acid β-oxidation axis, highlighting its potential as a biomarker and therapeutic target in HCC.

HCC↗

PubMed2006

Phenotypic abnormalities observed in aged cloned mice from embryonic stem cells after long-term maintenance.

Somatic/embryonic stem cell cloning has made it possible to produce an individual genomically identical to another individual. However, the cloned animals have a variety of abnormalities caused by the aberrant gene modification, with insufficient reprogramming in cloning. We previously reported abnormalities in cloned mice at birth. In this study, we examined what abnormalities could be seen in cloned mice after long-term maintenance. The aged cloned mice showed multiple abnormalities: increase of body weight, some phenotypic abnormalities in the kidneys, testes and thymus, and lower urea nitrogen in their serum biochemical values. The kidneys of all cloned mice were hypertrophied, with a metamorphic or whitish appearance. The multiple lesions, including the enlarged renal pelvis and distension of the renal veins in histology, might be the result of urine accumulation by urinary tract obstruction. The testes of the cloned mice were atrophied, and showed no sperm formation in histology. In contrast, the thymus was rather hypertrophied, and a comparably increased number of lymphocytes were observed in the medulla, consisting mainly of T cells. By conducting a progeny test between the cloned mice, it was confirmed that these abnormalities in the aged cloned mice were not transmitted to their offspring, indicating that the incomplete reprogramming in clones might be in part responsible for the abnormalities detected in aged clones. These results indicate that the postnatal abnormalities observed in aged cloned mice are varied and can be restored through the germ line.

Aging↗

PubMed2006

Gene expression profiling of individual bovine nuclear transfer blastocysts.

During somatic cell nuclear transfer the gene expression profile of the donor cell has to be changed or reprogrammed extensively to reflect that of a normal embryo. In this study we focused on the switching on of embryonic genes by screening with a microarray consisting of 5000 independent cDNA isolates derived from a bovine blastocyst library which we constructed for this purpose. Expression profiling was performed using linearly amplified RNA from individual day 7 nuclear transfer (NT) and genetically half-identical in vitro produced (IVP) blastocysts. We identified 92 genes expressed at lower levels in NT embryos whereas transcripts of 43 genes were more abundant in NT embryos (P < or = 0.05, > or = 1.5-fold change). A range of functional categories was represented among the identified genes, with a preponderance of constitutively expressed genes required for the maintenance of basal cellular function. Using a stringent quantitative SYBR-green real time RT-PCR based approach we found, when comparing the means of the expression levels of a larger set of individual embryos, that differences were small (< 2-fold) and only significant for two of the seven analysed genes (KRT18, SLC16A1). Notably, examination of transcript levels of a single gene in individual embryos could not distinguish an NT from a control embryo. This unpredictability of individual gene expression on a global background of multiple gene expression changes argues for a predominantly stochastic nature of reprogramming errors.

Animals↗

PubMedDate not supplied

Cell extract-derived differentiation of embryonic stem cells.

Various means have been used to encourage the differentiation of embryonic stem cells (ESCs) toward specific lineages, including growth factor administration, genetic modification, and coculture with relevant cells/tissues. Cell extract-based reprogramming has recently been used to derive mature cells from nonrelated phenotypes. In this communication, we tested whether this in vitro reprogramming approach can be used to direct ESC differentiation. Permeabilized murine ESCs exposed to extracts of murine type II pneumocytes showed increased expression of surfactant protein C and its corresponding mRNA, reflecting enhanced differentiation of pneumocytes. Subsequent differentiation to a type I phenotype was demonstrated by expression of aquaporin 5. Pneumocyte formation occurred quicker than with growth factor-induced differentiation. Our findings establish that ESCs can be differentiated in vitro using cellular extracts. This model provides a tool for analysis of the key factors involved in the differentiation of ESCs to type II pneumocytes.

Animals↗

PubMed1972

Programming generalization and maintenance of treatment effects across time and across settings.

Effects of three experimental and one control strategy were investigated in facilitating generalization and maintenance of treatment effects after two months in a token-economy classroom. At the conclusion of treatment, subjects were randomly assigned to one of three maintenance strategies or a control group and returned to their regular classrooms. The maintenance strategies were peer reprogramming, equating stimulus conditions between the experimental and regular classrooms, and teacher training in behavior management techniques. The maintenance strategies were implemented in the regular classroom for a two-month period and then terminated. Results indicated a powerful treatment effect produced by the token economy. Behavior maintenance effects following treatment were also obtained. The mean per cent appropriate behavior for the peer reprogramming and equating stimulus conditions strategies was significantly greater than the mean for the control subjects. The teacher training and control group means were not significantly different.

Journal Article↗

PubMed2006

[Lymphocyte trafficking and immunesurveillance].

The homeostasis of the immune system is maintained by the recirculation of naive lymphocytes through the secondary lymphoid tissues, such as the lymph nodes, Peyer's patches and spleen. Upon antigen encounter in the secondary lymphoid tissues, lymphocytes become activated and undergo a reprogramming of their trafficking properties. Most antigen-experienced lymphocytes traffic through the secondary lymphoid organs, but they can also migrate to extralymphoid tissues, where they exert effector functions. Dendritic cells in the secondary lymphoid tissues are crucial for the reprogramming of trafficking properties of activated T-lymphocytes. The exquisite specificity of such lymphocyte trafficking is determined by tissue-specific guidance signals expressed by the vascular endothelial cells, combined with counter receptors expressed by circulating lymphocytes. The high endothelial venules can selectively recruit naive lymphocytes into the lymph nodes and Peyer's patches by expressing a unique combination of vascular addressins and chemoattractants. The inflamed postcapillary venules in extralymphoid tissues also use a distinct array of endothelial adhesion molecules and tissue selective chemokines to support the recruitment of effector and memory lymphocytes that express appropriate trafficking receptors. Exit of lymphocytes from lymphoid and extralymphoid tissues into circulation is actively regulated by signals through specific receptors for sphingosine-1-phosphate and a certain chemokine(s), respectively. This review summarizes the present understandings of the mechanisms regulating homeostatic recirculation of naive lymphocytes through the secondary lymphoid tissues and tissue-specific trafficking of antigen-experienced lymphocytes.

Animals↗

PubMed2026

Spatial niche remodeling of senescent liver-resident immune cells and its role in chronic liver diseases.

The liver serves the triple functions of metabolism, detoxification, and immune surveillance. Its unique immune microenvironment is shaped by continuous exposure to gut-derived antigens, pathogen-associated molecular patterns (PAMPs), and metabolites arriving via the portal vein, necessitating a delicate equilibrium between immune tolerance and effector activation. This equilibrium relies on the coordinated activities of diverse liver-resident immune cell populations-including Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), dendritic cells (DCs), tissue-resident memory T cells (TRM), innate-like T cells, including mucosal-associated invariant T (MAIT) cells, natural killer T (NKT) cells, and γδ T cells, innate lymphoid cells (ILCs, encompassing conventional NK cells and helper ILC subsets), and neutrophils. With advancing age and chronic injury, these resident immune cell populations undergo profound senescence-associated phenotypic reprogramming that is spatially organized along the portal-to-central axis of the hepatic lobule. Key mechanisms include: telomere dysfunction and DNA damage accumulation driving persistent activation of p53/p21 and p16/Rb pathways; mitochondrial dysfunction with mitochondrial DNA (mtDNA) leakage fueling the senescence-associated secretory phenotype (SASP) via the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway; epigenetic age acceleration, including genome-wide H3K27me3 heterochromatinization; and metabolic reprogramming toward glycolysis and lipid accumulation. This review proposes a "spatial niche remodeling" framework to integrate these cell-intrinsic senescence programs with their lobular context, intercellular communication network rewiring, and pathogenic roles across the spectrum of chronic liver disease-from steatosis through steatohepatitis, fibrosis, cirrhosis, to hepatocellular carcinoma. We critically evaluate emerging senotherapeutic strategies targeting specific liver-resident immune cell subsets, discuss the barriers to clinical translation, and identify priority areas for future investigation, including the application of spatial multi-omics, humanized models, and epigenetic clock-guided clinical trials.

Kupffer cells↗

PubMed2026

Molecular mechanisms underlying drug resistance in protozoan parasites: emerging mechanisms and therapeutic perspectives.

Protozoan parasitic infections, including malaria, leishmaniasis, and human African trypanosomiasis, remain major global public health challenges. In the absence of highly effective vaccines, disease control relies primarily on chemotherapy; however, the emergence and spread of drug-resistant parasite populations increasingly threaten treatment efficacy. This review synthesizes current evidence on the molecular mechanisms underlying drug resistance in Plasmodium, Leishmania, and Trypanosoma species through a systematic analysis of literature. The review identifies four interconnected mechanisms that drive the evolution of drug resistance. First, altered drug transport enables parasites to regulate intracellular drug concentrations through mutations, loss, or amplification of membrane transporters, including PfCRT in Plasmodium and AQP2 in Trypanosoma brucei. Second, target modification and genomic plasticity promote resistance through point mutations in drug targets, such as dhfr and dhps in Plasmodium, while kinetoplastids, particularly Leishmania, exploit extensive genomic plasticity, including aneuploidy, gene amplification, and translational reprogramming, to facilitate rapid adaptation under drug pressure. Third, metabolic reprogramming enhances parasite survival by increasing intracellular thiol production, strengthening antioxidant defense systems, and reshaping central carbon and lipid metabolism to mitigate drug-induced stress. Finally, stress response and persistence mechanisms enable subpopulations of parasites to enter dormant, persister-like states characterized by reduced metabolic activity and slowed proliferation, thereby evading both host immune responses and chemotherapeutic agents. Collectively, these findings demonstrate that drug resistance is a dynamic, multifactorial evolutionary process rather than a single molecular event. Addressing this growing challenge requires integrating genomic surveillance, molecular diagnostics, mathematical modeling of resistance transmission, and mechanistic insights into parasite persistence into future drug discovery and disease control strategies. Such an integrated approach is essential for improving the durability of antiprotozoal therapies and advancing global efforts to control neglected protozoan diseases.

antiprotozoal therapy↗

PubMed2026

Exogenous ABA enhances cold tolerance of Rhododendron yedoense var. poukhanense under subzero temperature: integrating physiology, transcriptome, and proteome.

Low temperature limits the growth and ornamental value of evergreen shrubs. Rhododendron yedoense var. poukhanense, an important ornamental shrub from Northeast China, frequently suffers freezing damage during winter. While exogenous abscisic acid (ABA) enhances cold tolerance in many plants, its molecular mechanisms at subzero temperatures remain poorly understood in non-model species lacking chromosome-level reference genomes. This study investigated the effects of exogenous ABA on freezing tolerance in R. yedoense var. poukhanense at -4 °C using an integrated physiological, transcriptomic, and proteomic approach. Cutting seedlings were subjected to four treatments: CK (22°C control), A (22°C + ABA), LT (-4°C), and ALT (-4°C + ABA). Photosynthetic pigments, osmotic regulation substances, antioxidant enzyme activities, and malondialdehyde (MDA) content were measured. Transcriptome sequencing and quantitative proteomics were performed, and transcriptome data were validated by quantitative real-time PCR (qRT-PCR) of 15 selected genes. ABA pretreatment reduced visible cold injury severity, partially preserved photosynthetic pigments, decreased MDA content by 28.7%, and promoted recovery of catalase (+43.6%), superoxide dismutase (+31.1%), and peroxidase (+20.0%) activities under freezing stress. Transcriptome analysis revealed 8, 444 differentially expressed genes (DEGs) in LT versus CK and 6, 481 DEGs in ALT versus CK, representing a 23% reduction in transcriptional reprogramming scope attributable to ABA priming. The ALT versus LT comparison identified only 1, 690 additional DEGs, indicating that most cold-responsive genes were pre-activated during the ABA priming phase. Proteome analysis identified 1, 461 differentially expressed proteins (DEPs) in ALT versus CK. Integrated analysis revealed extensive post-transcriptional regulation, with transcript-protein concordance of only 1.0-4.1%, and co-enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in both omics layers. qRT-PCR validation confirmed high reliability of the transcriptome data (R2 = 0.8500). These findings demonstrate that exogenous ABA enhances freezing tolerance through multi-layered molecular regulation encompassing transcriptional buffering, translational reprogramming, and functional reallocation from photosynthesis to stress protection. This study provides the first integrated physiology-transcriptome-proteome framework for ABA-mediated freezing tolerance in an evergreen ornamental shrub and offers theoretical support for ABA-based winter protection strategies.

Rhododendron yedoense var. Poukhanense↗

PubMed2007

Alteration of the migratory behavior of UV-induced regulatory T cells by tissue-specific dendritic cells.

UV radiation-induced regulatory T cells (UV-Treg) inhibit the sensitization but not the elicitation of contact hypersensitivity when injected i.v. Because UV-Treg express the lymph node homing receptor CD62 ligand, upon i.v. injection they migrate into the lymph nodes but not into the periphery and therefore inhibit sensitization but not elicitation. We tried to modify the migratory behavior of UV-Treg with the aim to get them into the periphery and thereby to suppress the effector phase of immune reactions. Because the tissue selective homing of T effector cells is determined by tissue-specific dendritic cells (DC), we attempted to reprogram the migratory behavior of UV-Treg by DC. 2,4-Dinitrofluorobencene (DNFB)-specific UV-Treg coincubated with epidermal Langerhans cells (LC) blocked the elicitation upon i.v. injection into DNFB-sensitized mice. In contrast, i.v. injection of UV-Treg not incubated with LC did not inhibit the ear challenge. The same negative effect was observed for UV-Treg coincubated with DC from bone marrow, spleen, or lymph nodes. This effect was not due to different maturation stages as checked by MHC class II expression of the different DC types. Incubation with LC but not with bone marrow-derived DC down-regulated the expression of CD62 ligand on UV-Treg. Accordingly, CFDA-SE labeled UV-Treg coincubated with LC were found in the ears but not in the lymph nodes upon i.v. injection. This finding shows that the migratory behavior can be reprogrammed by tissue-specific DC and may have input on strategies trying to use Treg not only for the prevention but also for the treatment of immune-mediated diseases.

Animals↗

PubMed2002

Unintentional deactivation of implantable cardioverter-defibrillators in health care settings.

Patients with implanted pacemakers and defibrillators are routinely cautioned regarding exposure to environmental magnetic fields because such exposure may interfere with device function. Previous reports have confirmed interference with bingo wands, stereo speakers, and various workplace sources. In the 4 patients in this report, we document inadvertent alteration of the tachyarrhythmia detection function of implantable cardioverter-defibrillators (ICDs) that occurred in health care settings because of deliberately applied magnetic fields. Three of these patients had pectorally implanted ICDs that may have been confused with pacemakers, and 2 patients had undergone office surgical procedures at which time a magnet had been applied over the device. These events stemmed from (1) potential confusion by health care workers about the nature of the implanted device and (2) unique features in a specific manufacturer's defibrillator. We recommend the following steps to avoid such problems: (1) when device programming hardware and trained personnel are readily available, the patient's device should be interrogated and reprogrammed before and after any procedure involving electrocautery; (2) patients with ICDs should be monitored during device inactivation because they are unprotected from potentially life-threatening arrhythmias during this period; and (3) if the clinical situation does not allow device interrogation and reprogramming, the patient should be monitored electrocardiographically during magnet application and the device interrogated as soon as possible after magnet removal.

Aged↗

PubMed1985

Cardiac pacing practices: a survey.

A profile of permanent cardiac pacing practices in the United States during 1981 has been compiled in a survey of physicians who implant pacemakers. Approximately 5,600 physicians performed about 118,000 new primary implants at 3,670 centers. Only 17% of implantation procedures were replacements, compared with 31% in 1978. Although 90% of primary pacemakers were somewhat programmable, almost half were not reprogrammed within the first three months after implantation and 30% were never reprogrammed. Most patients (85%) were followed by trans-telephonic monitoring, 68% in conjunction with private office visits. The respondents estimated that dual-chamber pacing, accounting for 10% of implants in 1981, would increase to 37% by 1985.

Cardiac Pacing, Artificial↗

PubMed1999

[Use of dual-chamber pacemaker. Short and long-term results].

BACKGROUND: Continuous improvement of dual chamber DDD pacemakers, electrode stability and programmed sequential stimulation changed the prognosis of patients implanted with these devices. AIM: To report our experience with the use of dual chamber pacemakers. MATERIAL AND METHODS: One hundred seventy six patients (116 male), aged 13 to 91 years old, who received a dual chamber pacemaker implant, are reported. Patients were followed for a mean of 2.6 years. RESULTS: Indications for DDD pacemaker were complete atrioventricular block in 43%, sick sinus syndrome in 32%, paroxysmal A-V block in 24%. All pacemakers were Siemens-Pacesetter and were provided with an automatic sensing and threshold device. J shaped atrium electrodes were used in 78% of patients and screw-in electrodes in 22%. Post operative complications were displacement of atrial electrode in 8 patients, of ventricular electrode in 6 patients, infection in two patients and a hematoma in one. Chronic parameters, measured after six months, were within expected ranges and allowed a good reprogramming of pacemakers. Long term programming aimed to reduce battery depletion, enhance device performance and improve hemodynamic conditions. Normal sequential stimulation was achieved in 154 patients (87%), 14 (85) patients died of cardiovascular disease not related to pacemaker function. Eight patients were in atrial fibrillation and were reprogrammed to VVI and DDI modes. CONCLUSIONS: DDD pacemakers are reliable and afford symptomatic relief in a broad spectrum of patients.

Adolescent↗

PubMed1999

Superoxide attenuates macrophage apoptosis by NF-kappa B and AP-1 activation that promotes cyclooxygenase-2 expression.

Macrophages are a major source of cytokines and proinflammatory radicals such as superoxide. These mediators can be both produced and utilized by macrophages in autocrine-regulatory pathways. Therefore, we studied the potential role of oxygen radical-regulatory mechanisms in reprogramming macrophage apoptosis. Preactivation of RAW 264.7 cells with a nontoxic dose of the redox cycler 2,3-dimethoxy-1,4-naphthoquinone (5 microM) for 15 h attenuated S-nitrosoglutathione (1 mM)-initiated apoptotic cell death and averted accumulation of the tumor suppressor p53, which is indicative for macrophage apoptosis. Preactivation with superoxide promoted cyclooxygenase-2 induction that was NF-kappa B and AP-1 mediated. NF-kappa B activation was confirmed by p50/p65-heterodimer formation, I kappa B-alpha degradation, and stimulation of a NF-kappa B luciferase reporter construct. Furthermore, a NF-kappa B decoy approach abrogated cyclooxygenase-2 (Cox-2) expression as well as inducible protection. The importance of AP-1 for superoxide-mediated Cox-2 expression and cell protection was substantiated by using the extracellular signal-regulated kinase-inhibitor PD98059 and the p38-inhibitor SB203580, which blocked Cox-2 expression. In corroboration, Cox-2 expression was hindered by a dominant-negative c-jun mutant (TAM67). Protection from apoptosis was verified in human macrophages with the notion that superoxide promoted Cox-2 expression, which in turn attenuated nitric oxide-evoked caspase activation. We conclude that the sublethal generation of oxygen radicals reprograms macrophages by NF-kappa B and AP-1 activation. The resulting hyporesponsiveness reveals an attenuated apoptotic program in association with Cox-2 expression.

Adjuvants, Immunologic↗

PubMedDate not supplied

Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocysts.

We have established mouse embryonic stem (ES) cell lines from blastocysts derived by transfer of nuclei of fetal neuronal cells. These neuronal cell-derived embryonic cell lines had properties that characterize them as ES cells, including typical cell markers and alkaline phosphatase activity. Moreover, the cells had a normal karyotype and were pluripotent, as they were capable of differentiating into all three germ layers. Although they were derived from neuronal donor nuclei, the cells no longer expressed neuronal markers; however, they were capable of differentiating into cells with neuronal characteristics. These results suggest that the clone-derived cells have fully acquired an ES cell character. Thus, ES cells can be derived from embryos resulting from nuclear transfer, which results in reprogramming of the genetic information and acquisition of pluripotency. ES cells established from somatic cell-derived blastocysts could be useful not only as research tools for studying reprogramming but also as models for cell-based transplantation therapy.

Alkaline Phosphatase↗

PubMed2003

Mouse embryos cloned from brain tumors.

Cancer cells escape from growth control by accumulating genetic and epigenetic alterations. In rare instances, epigenetic changes alone are oncogenic. Furthermore, agents that modify DNA methylation or chromatin structure can restore a normal phenotype to cells harboring oncogenic mutations. However, it is unclear to what extent epigenetic reprogramming can reverse oncogenesis. Using somatic nuclear transfer, we show that medulloblastomas arising in Ptc1+/- mice can direct preimplantation development. Additionally, blastocysts derived from medulloblastoma nuclei form postimplantation embryos with typical cell layers. Thus, tumor cells can be epigenetically reprogrammed into normal cell types. This approach could lead to a general strategy for assessing genetic and epigenetic contributions to tumorigenesis.

Animals↗

PubMed2003

The effects of different donor cells and passages on development of reconstructed embryos.

In order to study the effects of different donor cells and passages on development of nuclear transfer embryos, we constructed embryos by electrofusing several kinds of donor cells into enucleated M II oocytes from Kun Ming (KM) mouse. These cells include 2-cell embryonic blastomeres, KMW embryonic stem (ES) cells, fetal fibroblast, ear fibroblast, tail tip fibroblast, sertoil cells and spermatogonia. Meanwhile, we compared the effects of passage numbers of fetal fibroblast cells on developmental competency after nuclear transfer. We found that 7.4% of reconstructed embryos from 2-cell embryonic blastomeres and 0.7% from ES cell could develop to blastocyst in vitro; embryos from fetal fibroblast could only develop to morula stage with the rate of 0.2%; embryos from spermatogonia could only develop to 8-cell stage and the rate was 0.3%; embryos respectively from ear fibroblast, sertoli cell and tail tip fibroblast could only develop to 4-cell stage. Although 2-cell development rate of embryos reconstructed from fetal fibroblast in first passage was significantly lower than those from the 2nd, the 3rd and the 4th passage, embryos from different passages could develop to 8-cell stage except the 3rd passage. The result indicated that it is more difficult for terminally differentiated cell nuclei to be reprogrammed in enucleated M II oocytes than for low differentiated cell nuclei. The reason of low development rate from ES cells maybe that most of ES cells was at S stage of the cell cycle, which out of coordination with M II oocytes. We could conclude that culture and passage of donor cells might be benefit to nucleus reprogramming.

Animals↗

PubMed2004

Establishment of life-span extended bovine fibroblast cells carrying the characterization of primary cells.

Although primary bovine embryonic fibroblast (BEF) cells have previously been used as nucleus-donors for nuclear transfer (NT), it has now been proposed to use BEF cells to generate cloned cows that were genetically modified by transgenic or a knock-out system. A major limitation to gene targeting somatic cells, however, is the overall life-span of the cell. In this study, we first examined in vitro life-span of primary BEF cells. Primary BEF cells were found to be replicative senescent at passage 10th-12th, similar to primary murine embryonic fibroblast cells. To overcome this short in vitro life-span, we have optimized culture conditions to extend the life-span and determined growth characteristics of BEF cell lines. Two life-span extended BEF cell lines (designated CGFR -BO-1 and CGFR-BO-2) were shown to grow much faster than their parental primary counterparts. Both cell lines did not display any potential for abnormal growth such as foci formations in either soft-agar or confluent culture condition. In cloning experiments using these cell lines as a nuclear donor, the reconstructed karyoblasts underwent apoptosis, reprogramming and development in the blastocyst stage, at a similar frequency to those observed with parental as well as adult primary fibroblasts. Furthermore, these cell lines targeted with green fluorescence protein (GFP) were successfully transduced, selected and reprogrammed by NT to develop into a blastocyst stage with GFP expression. Our results suggested methods to extend life-span of donor cells with tremendous implications for the genetic engineering of bovine fibroblast cells.

Animals↗