scholarly journals The Effect of the Expression of the Antiapoptotic BHRF1 Gene on the Metabolic Behavior of a Hybridoma Cell Line

2021 ◽  
Vol 11 (14) ◽  
pp. 6258
Author(s):  
Iván Martínez-Monge ◽  
Pere Comas ◽  
David Catalán-Tatjer ◽  
Jordi Prat ◽  
Antoni Casablancas ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Lactate Production ◽  
Culture Broth ◽  
Cell Physiology ◽  
Nadh Regeneration ◽  
General Terms ◽  
Early Protein ◽  
Glutamine Consumption

One of the most important limitations of mammalian cells-based bioprocesses, and particularly hybridoma cell lines, is the accelerated metabolism related to glucose and glutamine consumption. The high uptake rates of glucose and glutamine (i.e., the main sources of carbon, nitrogen and energy) lead to the production and accumulation of large amounts of lactate and ammonia in culture broth. Lactate and/or ammonia accumulation, together with the depletion of the main nutrients, are the major causes of apoptosis in hybridoma cell cultures. The KB26.5 hybridoma cell line, producing an IgG3, was engineered with BHRF1 (KB26.5-BHRF1), an Epstein–Barr virus-encoded early protein homologous to the antiapoptotic protein Bcl-2, with the aim of protecting the hybridoma cell line from apoptosis. Surprisingly, besides achieving effective protection from apoptosis, the expression of BHRF1 modified the metabolism of the hybridoma cell line. Cell physiology and metabolism analyses of the original KB26.5 and KB26.5-BHRF1 revealed an increase of cell growth rate, a reduction of glucose and glutamine consumption, as well as a decrease in lactate secretion in KB26.5-BHRF1 cells. A flux balance analysis allowed us to quantify the intracellular fluxes of both cell lines. The main metabolic differences were identified in glucose consumption and, consequently, the production of lactate. The lactate production flux was reduced by 60%, since the need for NADH regeneration in the cytoplasm decreased due to a more than 50% reduction in glucose uptake. In general terms, the BHRF1 engineered cell line showed a more efficient metabolism, with an increase in biomass volumetric productivity under identical culture conditions.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

scholarly journals Cell Line Classification Using Electric Cell-Substrate Impedance Sensing (ECIS)

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Megan L. Gelsinger ◽  
Laura L. Tupper ◽  
David S. Matteson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Statistical Tests ◽  
Classification Methods ◽  
Impedance Sensing ◽  
Mammalian Cell Lines ◽  
Cell Substrate ◽  
Multiple Cell ◽  
Line Classification

AbstractWe present new methods for cell line classification using multivariate time series bioimpedance data obtained from electric cell-substrate impedance sensing (ECIS) technology. The ECIS technology, which monitors the attachment and spreading of mammalian cells in real time through the collection of electrical impedance data, has historically been used to study one cell line at a time. However, we show that if applied to data from multiple cell lines, ECIS can be used to classify unknown or potentially mislabeled cells, factors which have previously been associated with the reproducibility crisis in the biological literature. We assess a range of approaches to this new problem, testing different classification methods and deriving a dictionary of 29 features to characterize ECIS data. Most notably, our analysis enriches the current field by making use of simultaneous multi-frequency ECIS data, where previous studies have focused on only one frequency; using classification methods to distinguish multiple cell lines, rather than simple statistical tests that compare only two cell lines; and assessing a range of features derived from ECIS data based on their classification performance. In classification tests on fifteen mammalian cell lines, we obtain very high out-of-sample predictive accuracy. These preliminary findings provide a baseline for future large-scale studies in this field.

scholarly journals Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line.

1984 ◽  
Vol 4 (10) ◽  
pp. 2214-2223 ◽  
Author(s):  
K T Jeang ◽  
M S Cho ◽  
G S Hayward
Keyword(s):  
Cell Line ◽  
Mammalian Cells ◽  
Regulatory Region ◽  
Constitutive Expression ◽  
Regulatory Elements ◽  
Mouse Cell ◽  
Cellular Protein ◽  
Immediate Early ◽  
Early Protein ◽  
Infected Cells

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.

scholarly journals Targeted Comparative RNA Interference Analysis Reveals Differential Requirement of Genes Essential for Cell Proliferation

2006 ◽  
Vol 17 (11) ◽  
pp. 4837-4845 ◽  
Author(s):  
Yuichi J. Machida ◽  
Yuefeng Chen ◽  
Yuka Machida ◽  
Ankit Malhotra ◽  
Sukumar Sarkar ◽  
...  
Keyword(s):  
Rna Interference ◽  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Ribosome Biogenesis ◽  
Cancer Cell Line ◽  
Prostate Cancer Cell Line ◽  
Breast Epithelial Cell ◽  
Epithelial Cell Line ◽  
Dependent Manner

Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 growth-regulated genes in parallel in a breast epithelial cell line, MCF10A, and a prostate cancer cell line, PC3. Depletion of an unexpectedly high number of genes (25%) differentially affected proliferation of the two cell lines. Knockdown of many genes that spare PC3 (p53−) but inhibit MCF10A (p53+) proliferation induces p53 in MCF10A cells. EBNA1BP2, involved in ribosome biogenesis, is an example of such a gene, with its depletion arresting MCF10A at G1/S in a p53-dependent manner. TARCOR is thus useful for identifying cell type–specific genes and pathways involved in proliferation and also for exploring the heterogeneity of cell lines. In particular, our data emphasize the importance of considering the genetic status, when performing siRNA screens in mammalian cells.

NanoLiterBioReactor: Monitoring of Long-Term Mammalian Cell Physiology at Nanofabricated Scale

2004 ◽  
Vol 820 ◽  
Author(s):  
Ales Prokop ◽  
Zdenka Prokop ◽  
David Schaffer ◽  
Eugene Kozlov ◽  
John Wikswo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Cell Counting ◽  
Cell Physiology ◽  
Mammalian Cell Lines ◽  
Cell Counts ◽  
Multiple Cell

AbstractThere is a need for microminiaturized cell-culture environments, i.e., NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. Reduced NBR volumes would not only shorten the time required for diffusive mixing, for achieving thermal equilibrium, and for cells to grow to confluence, but also simplify accurate cell counting, minimize required volumes of expensive analytical pharmaceuticals or toxins, and allow for thousands of culture chambers on a single instrumented chip. These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. The challenge, beyond that of optimizing the NBR physically, is to detect cellular response, provide appropriate control signals, and, eventually, facilitate closed-loop adjustments of the environment--e.g., to control temperature, pH, ionic concentration, etc., to maintain homeostasis, or to apply drugs or toxins followed by the adaptive administration of a selective toxin antidote. To characterize in a nonspecific manner the metabolic activity of cells, the biosensor elements of the NBR might include planar pH, dissolved oxygen, and redox potential sensors, or even an isothermal picocalorimeter (pC) to monitor thermodynamic response. Equipped with such sensors, the NBR could be used to perform short- and long-term cultivation of several mammalian cell lines in a perfused system, and to monitor their response to analytes in a massively parallel format. This approach will enable automated, parallel, and multiphasic monitoring of multiple cell lines for drug and toxicology screening. An added bonus is the possibility of studying cell populations with low cell counts whose constituents are completely detached from typical tissue environment, or populations in controlled physical and chemical gradients.

scholarly journals Cultivation of potato leafroll virus (PLRV) in mammalian continuous cell lines

2018 ◽  
Vol 5 (3) ◽  
pp. 19-26
Author(s):  
I. Volkova ◽  
L. Reshotko ◽  
T. Bova ◽  
O. Dmytruk ◽  
S. Derevianko
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Neutralization Test ◽  
Potato Leafroll Virus ◽  
Continuous Cell Line ◽  
Rabbit Kidney ◽  
Continuous Cell Lines ◽  
Physical Chemical ◽  
Leafroll Virus

Aim. To use the ability of potato leafroll virus (PLRV) to infect and multiply in mammalian continuous cell lines to purify PLRV isolates from the vegetative plant material, and to study the pathogenicity of those isolates for plants (after culturing in mammalian continuous cell line), to investigate morphological, physical-chemical, biological and antigen properties of PLRV isolates from mammalian cells and to study an alternative diagnostic method – the neutralization test in the mammalian continuous cell lines. Methods. The methods of cultivating animal viruses in the mammalian continuous cell line, microscopical biochemical, and serological methods, the method of artifi cial nutrition of aphids are detailed under Material and Methods. Results. It was demonstrated that successful cultivation of PLRV in mammalian continuous cell line allowed obtaining pure virus isolates from potato plants and aphids and preserving them for a long time (over a period of 7 years). The cultivation of PLRV in the mammalian continuous cell line did not impact its pathogenic properties and allowed transmitting the virus to plants. Continuous cells lines of pig embryonic kidney (PEKV), of kidney Syrian hamster (BHK- 21), of testicles of piglets (PTP), of kidneys of the bull (MDBC), and of carcinoma rabbit kidney (RK-13) were found to be sensitive to PLRV, Con tinuous cell lines of human (HeLa, Hep-2 and of African green monkey kidney (Vero) were not infected by the virus. The infectious activity of PLRV in the sensitive continuous cell lines was 20–8.5 lg TCD 50 /ml depending on the cell line. The isolates of PLRV were resistant to lipid- dissolving solvents, multiplied in a pH range from 4.0 till 10.0 and were thermoresistant at 50 oС in the absence of bivalent ions of magnesium, ТIP was in the range of 60–65 oС under our experimental conditions. The optimal temperature for the reproduction of PLRV in the cell culture was c. 24 °С. The use of neutralization test in the mammalian continuous cell line allowed isolation in pure culture and identifi cation of PLRV reliably in a time span of c. 14 days. Conclusions. It was proven that PLRV can be cultivated in the mammalian continuous cell lines of PEKV, ВНК-21, PTV, MDВК and RK-13. It was established that the cultivation of PLRV in these continuous cell lines did not impact its biological, pathogenic, antigenic and physical-chemical properties. The identifi cation of pure cultures of PLRV obtained in mammalian cells can be reliably performed by the use of neutralization reaction.

scholarly journals Evolution from adherent to suspension: systems biology of HEK293 cell line development

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Magdalena Malm ◽  
Rasool Saghaleyni ◽  
Magnus Lundqvist ◽  
Marco Giudici ◽  
Veronique Chotteau ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Viral Vectors ◽  
Hek293 Cell ◽  
Cholesterol Biosynthesis ◽  
Human Cell Line ◽  
Gene Copy ◽  
Cell Line Development ◽  
Suspension Systems

Abstract The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

NanoLiterBioReactor: Monitoring of Long-Term Mammalian Cell Physiology at Nanofabricated Scale

2004 ◽  
Vol 823 ◽  
Author(s):  
Ales Prokop ◽  
Zdenka Prokop ◽  
David Schaffer ◽  
Eugene Kozlov ◽  
John Wikswo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Cell Counting ◽  
Cell Physiology ◽  
Mammalian Cell Lines ◽  
Cell Counts ◽  
Multiple Cell

AbstractThere is a need for microminiaturized cell-culture environments, i.e., NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. Reduced NBR volumes would not only shorten the time required for diffusive mixing, for achieving thermal equilibrium, and for cells to grow to confluence, but also simplify accurate cell counting, minimize required volumes of expensive analytical pharmaceuticals or toxins, and allow for thousands of culture chambers on a single instrumented chip. These devices would enable the development of a new class of miniature, automated cell- based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. The challenge, beyond that of optimizing the NBR physically, is to detect cellular response, provide appropriate control signals, and, eventually, facilitate closed-loop adjustments of the environment–e.g., to control temperature, pH, ionic concentration, etc., to maintain homeostasis, or to apply drugs or toxins followed by the adaptive administration of a selective toxin antidote. To characterize in a nonspecific manner the metabolic activity of cells, the biosensor elements of the NBR might include planar pH, dissolved oxygen, and redox potential sensors, or even an isothermal picocalorimeter (pC) to monitor thermodynamic response. Equipped with such sensors, the NBR could be used to perform short- and long-term cultivation of several mammalian cell lines in a perfused system, and to monitor their response to analytes in a massively parallel format. This approach will enable automated, parallel, and multiphasic monitoring of multiple cell lines for drug and toxicology screening. An added bonus is the possibility of studying cell populations with low cell counts whose constituents are completely detached from typical tissue environment, or populations in controlled physical and chemical gradients.

scholarly journals Fine-resolution analysis of products of intrachromosomal homeologous recombination in mammalian cells.

1997 ◽  
Vol 17 (7) ◽  
pp. 3614-3628 ◽  
Author(s):  
D Yang ◽  
A S Waldman
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Average Rate ◽  
Sequence Divergence ◽  
Insertion Mutant ◽  
Insertion Mutation ◽  
Sequence Identity ◽  
Homeologous Recombination ◽  
Hsv 1

Mouse Ltk- cell lines that contained a herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene with a 16-bp insertion mutation linked to either a defective HSV-2 tk gene or a hybrid tk sequence comprised of HSV-1 and HSV-2 tk sequences were constructed. HSV-1 and HSV-2 tk genes have 81% nucleotide identity and hence are homeologous. Correction of the insertion mutant HSV-1 tk gene via recombination with the hybrid tk sequence required an exchange between homeologous tk sequences, although recombination could initiate within a region of significant sequence identity. Seven cell lines containing linked HSV-1 and HSV-1-HSV-2 hybrid tk sequences gave rise to tk+ segregants at an average rate of 10(-8) events per cell division. DNA sequencing revealed that each recombinant from these lines displayed an apparent gene conversion which involved an accurate transfer of an uninterrupted block of information between homeologous tk sequences. Conversion tract lengths ranged from 35 to >330 bp. In contrast, cell lines containing linked HSV-1 and HSV-2 tk sequences with no significant stretches of sequence identity had an overall rate of homeologous recombination of <10(-9). One such cell line produced homeologous recombinants at a rate of 10(-8). Strikingly, all homeologous recombinants from this latter cell line were due to crossovers between the HSV-1 and HSV-2 tk genes. Our results, which provide the first detailed analysis of homeologous recombination within a mammalian genome, suggest that rearrangements in mammalian genomes are regulated by the degree of sequence divergence located at the site of recombination initiation.

scholarly journals Introduction and recovery of a selectable bacterial gene from the genome of mammalian cells

1982 ◽  
Vol 2 (8) ◽  
pp. 966-976
Author(s):  
M L Breitman ◽  
L C Tsui ◽  
M Buchwald ◽  
L Siminovitch
Keyword(s):  
Molecular Weight ◽  
Cell Line ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Dna Molecules ◽  
High Molecular Weight Dna ◽  
Bacterial Gene ◽  
E Coli ◽  
Transformed Cell ◽  
Transformed Cell Lines

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells.

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