Scaffold/matrix attachment regions from CHO cell chromosome enhanced the stable transfection efficiency and the expression of transgene in CHO cells

2014 ◽  
Vol 61 (5) ◽  
pp. 510-516 ◽  
Author(s):  
Ming Chang ◽  
Ruijie Liu ◽  
Qingzhe Jin ◽  
Yuanfa Liu ◽  
Xingguo Wang
Keyword(s):  
Cho Cells ◽  
Transfection Efficiency ◽  
Matrix Attachment Regions ◽  
Stable Transfection ◽  
Cho Cell ◽  
Cell Chromosome

scholarly journals Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia

2021 ◽  
Vol 22 (10) ◽  
pp. 5218
Author(s):  
Tomu Kamijo ◽  
Takahiro Kaido ◽  
Masahiro Yoda ◽  
Shinpei Arai ◽  
Kazuyoshi Yamauchi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cho Cells ◽  
Fibrinogen Level ◽  
Culture Media ◽  
Chinese Hamster ◽  
Plasma Fibrinogen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cho Cell ◽  
Accelerated Degradation ◽  
Heterozygous Patient

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients’ plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and “D:D” interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient’s hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

scholarly journals Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180 ◽  
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

scholarly journals Plasma Membrane Effects of Sphingolipid-Synthesis Inhibition by Myriocin in CHO Cells: A Biophysical and Lipidomic Study

2021 ◽  
Author(s):  
Bingen G. Monasterio ◽  
Noemi Jiménez-Rojo ◽  
Aritz B. García-Arribas ◽  
Howard Riezman ◽  
Félix M. Goñi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cho Cells ◽  
Plasma Membranes ◽  
Mechanical Resistance ◽  
Serine Palmitoyltransferase ◽  
Cho Cell ◽  
Cellular Effects ◽  
Inhibitory Compound ◽  
Membrane Effects ◽  
Biophysical Changes

Abstract Two main strategies for establishing the cellular effects of a given enzyme activity suppression are (a) the use of a stably mutated cell line that lacks a functional gene, or (b) treating the wild type with an inhibitory compound that affects the same gene-product protein. In this work, myriocin was used to block the serine palmitoyltransferase (SPT) enzyme of CHO cells and the subsequent biophysical changes in membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) happened to be lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated glycerophospholipids than the non-treated ones, although they achieved it only partially, their plasma membranes remaining more fluid and less penetrable than those from the control cells.

Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family

1990 ◽  
Vol 10 (10) ◽  
pp. 5160-5165
Author(s):  
S Ahmad ◽  
R Ahuja ◽  
T J Venner ◽  
R S Gupta
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Immunofluorescent Staining ◽  
Mutant Form ◽  
Two Dimensional ◽  
Wild Type ◽  
Cho Cell ◽  
Microtubule Inhibitors

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.

The AML Prognostic Marker, AF1q, Is Directly Regulated by MiR-29b.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2603-2603
Author(s):  
Yin Xiong ◽  
Zejuan Li ◽  
Aik Choon Tan ◽  
Judson Bemis ◽  
Xiuyan Xie ◽  
...  
Keyword(s):  
Cell Lines ◽  
Conflicts Of Interest ◽  
Prognostic Biomarker ◽  
Transfection Efficiency ◽  
Inverse Correlation ◽  
Underlying Mechanism ◽  
Fusion Partner ◽  
Stable Transfection ◽  
Elevated Expression ◽  
Normal Cytogenetics

Abstract Abstract 2603 Poster Board II-579 We have consistently shown that elevated expression of AF1q, an MLL fusion partner, is a poor prognostic biomarker for pediatric acute myeloid leukemia (AML), adult AML with normal cytogenetics (NC-AML), and adult myelodysplastic syndrome (MDS). However, the underlying mechanism of how AF1q is regulated in normal and abnormal hematopoiesis is still unclear. Our previous studies suggest that AF1q is highly regulated during hematopoietic cell differentiation and development and it is known that genes related to cell development and differentiation are likely to be regulated by various microRNAs. We used a variety of the web based programs to identify microRNA candidates that may potentially regulate AF1q based on the predicted targeting efficiency. We found the strongest predicted binder to the AF1q 3′untranslated region (3′UTR) was miR-29b, a member of the miR-29 family which has recently been characterized to regulate a member of the Bcl-2 family protein, Mcl-1 and other leukemia related oncogenes. We found that MiR-29b expression had a significantly inverse correlation (p<0.05) with AF1q expression in a cohort of more than 60 AML patients. This relationship is consistent with microRNA/AF1q regulation. In order to determine if miR-29b directly regulates AF1q, we chose H157 and SKMES1 lung cancer cell lines which were known to have high AF1q expression and high transfection efficiency to test if transfection of miR-29b into cells can suppress its endogenous AF1q expression. Our data showed that transfection of miR-29b into these two cell lines could indeed significantly suppress the AF1q expression both in H157 (p=0.001) and SKMES1 (p=0.004) cells. Then we wanted to determine if the miR-29b binding domain is specific in the AF1q 3′UTR region. Two reporter plasmids that contained the germline AF1q 3'UTR (GFP-A3U) and mutant AF1q 3′UTR in its microRNA binding site (GFP-A3U-Mutant) were constructed. We found that GFP readout was significantly suppressed in a stable transfection with native AF1q 3′UTR (GFP-A3U); in contrast, the GFP readout was not suppressed in a stable transfection with GFP-A3U-Mutant, suggesting that miR-29b is able to bind to the germline AF1q 3′UTR but not to the mutated 3′UTR (GFP-A3U-Mutant). These observations proved that miR-29b specifically regulates AF1q expression through its binding to the AF1q 3′UTR. Taking these observations together, we conclude that miR-29b directly regulates the leukemia associated gene AF1q. Elevated AF1q expression was found to have clinical significance in AML, thus, these findings warrant further investigation to determine if miR-29b will be able to serve as a prognostic biomarker for AML patients. Disclosures: No relevant conflicts of interest to declare.

B-domain deleted factor VIII is aggregated and degraded through proteasomal and lysosomal pathways

2005 ◽  
Vol 93 (05) ◽  
pp. 824-832 ◽  
Author(s):  
Benoit Guillet ◽  
Cécile Ducasse ◽  
Nathalie Enjolras ◽  
Marie-Hélène Rodriguez ◽  
Véronique Rolli ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Cho Cells ◽  
Mammalian Cells ◽  
Mrna Synthesis ◽  
Extracellular Production ◽  
Single Chain ◽  
Cho Cell ◽  
Over Expression ◽  
Triton X

SummaryFactor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60–80%) and insoluble fractions (for 20–40%). The incubation of the expressing cells with tunicamycin (5 μg/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH4Cl, leupeptin, clasto-Lactacystin β-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes.

scholarly journals Polyamine starvation prolongs the S and G2 phases of polyamine-dependent (arginase-deficient) CHO cells.

1984 ◽  
Vol 4 (5) ◽  
pp. 915-922 ◽  
Author(s):  
S Anehus ◽  
P Pohjanpelto ◽  
B Baldetorp ◽  
E Långström ◽  
O Heby
Keyword(s):  
Cell Cycle ◽  
Ornithine Decarboxylase ◽  
Cho Cells ◽  
Decarboxylase Activity ◽  
Serum Free Medium ◽  
Free Medium ◽  
Cho Cell ◽  
Irreversible Inhibitor ◽  
Serum Free ◽  
Cell Variant

This study analyzes the effects of polyamine starvation on cell cycle traverse of an arginase-deficient CHO cell variant (CHO-A7). These cells grow well in serum-free medium, provided that it contains ornithine or polyamines or both. In the absence of ornithine or polyamines or both, the CHO-A7 cells develop severe polyamine deficiency and, as a consequence, grow more slowly. When grown to a stationary phase in the presence of ornithine or putrescine or both, the CHO-A7 cells became arrested in G0/early G1. However, when starved for ornithine and polyamines, they accumulated in the S and G2 phases. Ornithine and polyamine starvation of CHO-A7 cells causes an increase in ornithine decarboxylase activity. When this increase was prevented by treatment with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, growth was further suppressed, and a greater fraction of cells were found in the S and G2 phases of the cell cycle.

Improved recombinant gene expression in CHO cells using matrix attachment regions

2004 ◽  
Vol 107 (2) ◽  
pp. 95-105 ◽  
Author(s):  
Jong-Mook Kim ◽  
Jung-Seob Kim ◽  
Doo-Hong Park ◽  
Ho Sung Kang ◽  
Jaeseung Yoon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cho Cells ◽  
Matrix Attachment Regions ◽  
Recombinant Gene Expression ◽  
Recombinant Gene

scholarly journals Cytopathogenic mechanisms of Entamoeba histolytica.

1980 ◽  
Vol 152 (2) ◽  
pp. 377-390 ◽  
Author(s):  
J I Ravdin ◽  
B Y Croft ◽  
R L Guerrant
Keyword(s):  
Entamoeba Histolytica ◽  
Cho Cells ◽  
Cytochalasin B ◽  
Chinese Hamster ◽  
Cytochalasin D ◽  
Target Cells ◽  
Cho Cell ◽  
The One ◽  
Dose Dependent ◽  
Microtubule Function

Cinemicrography of Entamoeba histolytica destruction of Chinese hamster ovary (CHO) cells shows that ameba cytopathogenicity consists of separate components: a contact-dependent cytolethal effect, and phagocytosis. Cells not in contact with amebae remain intact. Quantitation of ameba destruction of CHO cells by applying the one-hit hypothesis confirms that the cytoethal effect of amebae is contact dependent. Studies with 111Indium oxine-labeled cells provide further evidence of extracellular killing by E. histolytica and indicate that &gt; 94% of the target cells are killed before phagocytosis. When we examined for a cytotoxin release by E. histolytica, we found no effect on CHO cells with filtrates of amebae, and a nonspecific effect of cell rounding and release with sonicates of amebae. The ameba sonicate effect was time-dose dependent, was not cytolethal, was reversible, and was inhibited by alpha II macroglobulin. Cytochalasin B altered ameba motility and morphology, and monolayer experiments confirmed that cytochalasins A, B, or D inhibited CHO cell destruction by E. histolytica. Cytochalasin D also inhibited extracellular killing of CHO cells by amebae in pellets, apparently independent of effects on ameba motility or phagocytosis. Colchicine and vinblastine, alone or in combination with cytochalasin D, did not inhibit E. histolytica cytopathogenicity, which indicates that microtubule function is not required for target cell killing by amebae.

Sign in / Sign up

Export Citation Format

Share Document