scholarly journals TRANSFECTION OF EXOGENOUS DNA IN CHICKEN OVIDUCT CELLS IN VITRO

2015 ◽  
Vol 14 (1) ◽  
pp. 29-31
Author(s):  
A. V. Samoilov ◽  
N. M. Suraeva
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Primary Culture ◽  
Cultured Cells ◽  
Expression System ◽  
Enzyme Treatment ◽  
Exogenous Dna ◽  
Chicken Oviduct ◽  
Development In Vitro

Effective production of pharmaceutical proteins using transgenic poultry may be achieved only from a construction of expression vector for chicken oviduct bioreactor. This study was focused on development of temporary expression system by using primary oviduct epithelial cells in which transfected gene expression can be studied. The present work was aimed to study three manners for preparation of primary oviduct cells, but only one was effective. Selective digestion of chicken oviduct tissue pieces in trypsin at 38° C and mechanical withdrawal of upper cell layer resulted in the isolation of purity primary culture. Experiments indicated that monolayer cultured for 2-3 d were fit to be passaged. Then after 1-2-d, the cells were transfected. Based on our result with primary culture, we did not used complicated enzyme treatment and extra equipment in order to increase the purity of epithelial cells. Liposomes «Lipofectine® 2000» were used for primary oviduct cells transfection of a plasmid designed based on the pIRES EGFP2 vector (Clontech, United States). The ratio of cells carrying GFP activity was 5-8% of the total number of cultured cells. This researches was focused on development in vitro of temporary expression system in which transfected gene expression can be tested for 4-5 d.

The VSL#3 probiotic formula induces mucin gene expression and secretion in colonic epithelial cells

2007 ◽  
Vol 292 (1) ◽  
pp. G315-G322 ◽  
Author(s):  
C. Caballero-Franco ◽  
K. Keller ◽  
C. De Simone ◽  
K. Chadee
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
In Vivo Studies ◽  
Lactobacillus Species ◽  
Mucin Secretion ◽  
Colonic Epithelial Cells ◽  
Dnase Treatment

Several studies have stressed the importance of the microbiota in the maintenance of the gastrointestinal epithelium. Administration of probiotic bacteria, supplements composed of microbiota constituents, was previously shown to diminish symptoms in patients suffering from inflammatory bowel diseases. This raises the possibility that probiotics may play an active role in enhancing the intestinal barrier at the mucosal surface. In this study, we investigated whether the clinically tested VSL#3 probiotic formula and/or its secreted components can augment the protective mucus layer in vivo and in vitro. For in vivo studies, Wistar rats were orally administered the probiotic mixture VSL#3 on a daily basis for seven days. After treatment, basal luminal mucin content increased by 60%. In addition, we exposed isolated rat colonic loops to the VSL#3 probiotic formula, which significantly stimulated colonic mucin (MUC) secretion and MUC2 gene expression; however, MUC1 and MUC3 gene expression were only slightly elevated. The effect of the VSL#3 mucin secretagogue was also tested in vitro by use of LS 174T colonic epithelial cells. In contrast to the animal studies, cultured cells incubated with VSL#3 bacteria did not exhibit increased mucin secretion. However, the bacterial secreted products contained in the conditioned media stimulated a remarkable mucin secretion effect. Among the three bacterial groups ( Lactobacilli, Bifidobacteria, and Streptococci) contained in VSL#3, the Lactobacillus species were the strongest potentiator of mucin secretion in vitro. A preliminary characterization of the putative mucin secretagogue suggested that it was a heat-resistant soluble compound, which is not sensitive to protease and DNase treatment. These findings contribute to a better understanding of the complex and beneficial interaction between colonic epithelial cells and intestinal bacteria.

scholarly journals A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

2008 ◽  
Vol 22 (12) ◽  
pp. 2677-2688 ◽  
Author(s):  
Paul G. Tiffen ◽  
Nader Omidvar ◽  
Nuria Marquez-Almuina ◽  
Dawn Croston ◽  
Christine J. Watson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Oncostatin M ◽  
Specific Gene ◽  
Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

Cultured epithelial cells derived from human foetal pancreas as a model for the study of cystic fibrosis: further analyses on the origins and nature of the cell types

1988 ◽  
Vol 90 (1) ◽  
pp. 73-77
Author(s):  
A. Harris ◽  
L. Coleman
Keyword(s):  
Cystic Fibrosis ◽  
Tissue Culture ◽  
Epithelial Cells ◽  
Culture System ◽  
Cultured Cells ◽  
Cell Types ◽  
Cultured Epithelial Cells ◽  
Different Cell Types ◽  
Foetal Pancreas

The establishment of a tissue-culture system for epithelial cells derived from human foetal pancreas has recently been reported. Further analyses have now been made on these cells in vitro, together with parallel investigation of the distribution of different cell types within the intact foetal pancreas. Results support the view that the cultured cells are ductal in origin and nature. Pancreatic epithelial cell cultures have also been established from foetuses with cystic fibrosis.

scholarly journals Single cell RNA sequencing of calvarial and long bone endocortical cells

2019 ◽  
Author(s):  
Ugur M. Ayturk ◽  
Joseph P. Scollan ◽  
Alexander Vesprey ◽  
Christina M. Jacobsen ◽  
Paola Divieti Pajevic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Long Bone ◽  
Appendicular Skeleton ◽  
Rna Seq ◽  
Isolated Cells

ABSTRACTSingle cell RNA-seq (scRNA-seq) is emerging as a powerful technology to examine transcriptomes of individual cells. We determined whether scRNA-seq could be used to detect the effect of environmental and pharmacologic perturbations on osteoblasts. We began with a commonly used in vitro system in which freshly isolated neonatal mouse calvarial cells are expanded and induced to produce a mineralized matrix. We used scRNA-seq to compare the relative cell type abundances and the transcriptomes of freshly isolated cells to those that had been cultured for 12 days in vitro. We observed that the percentage of macrophage-like cells increased from 6% in freshly isolated calvarial cells to 34% in cultured cells. We also found that Bglap transcripts were abundant in freshly isolated osteoblasts but nearly undetectable in the cultured calvarial cells. Thus, scRNA-seq revealed significant differences between heterogeneity of cells in vivo and in vitro. We next performed scRNA-seq on freshly recovered long bone endocortical cells from mice that received either vehicle or Sclerostin-neutralizing antibody for 1 week. Bone anabolism-associated transcripts were also not significantly increased in immature and mature osteoblasts recovered from Sclerostin-neutralizing antibody treated mice; this is likely a consequence of being underpowered to detect modest changes in gene expression, since only 7% of the sequenced endocortical cells were osteoblasts, and a limited portion of their transcriptomes were sampled. We conclude that scRNA-seq can detect changes in cell abundance, identity, and gene expression in skeletally derived cells. In order to detect modest changes in osteoblast gene expression at the single cell level in the appendicular skeleton, larger numbers of osteoblasts from endocortical bone are required.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

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