Engineered plant cell or fungal association with bacteria that fix nitrogen.
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Bacteria↗
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Results for “Cell Engineering”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
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[Mitochondrial vectors in the gene engineering of cells of lower eukaryotes].
The possibility of using the eukaryotic mitochondrial DNA and mitochondrial plasmids as suitable shuttle vectors for pro- and eukaryotic systems is discussed. Data are presented on the expression of the animal mitochondrial DNA in bacterial and yeasts cells.
Animals↗
CERTOMICS: trusted single-cell multiomics pipeline for high-resolution profiling of adoptive cellular immunotherapies.
SUMMARY: Adoptive cellular immunontherapies, such as chimeric antigen receptor (CAR) T cell therapy, have transformed cancer treatment, yet challenges such as resistance, relapse, and high costs limit their efficacy and accessibility. A comprehensive understanding of cellular heterogeneity and molecular profiles is essential to improve these therapies. Advanced single-cell multiomics technologies have the power to analyze the complex interactions between CAR-engineered cells, immune cells, and tumor cells. However, standardized single-cell multiomics computational pipelines specifically tailored to CAR-engineered cell products are lacking. Due to the synthetic nature of CAR transgenes, additional steps for reliable identification and characterization of CAR-positive cells are required but not included in existing data-processing workflows. To address this, we present CERTOMICS, a Nextflow-based, CAR-aware pipeline offering enhanced CERTainty in immunophenotyping and data interpretation, tailored for single-cell multiOMICSprofiling of adoptive cellular immunotherapies. The pipeline standardizes processing 10x Genomics single-cell multiomics data and integrates CAR-specific identification and quality control. Additionally, a curated repository of CAR construct sequences and annotation data is provided, serving as an extensible resource to support the analysis and development of CAR T cell therapies. AVAILABILITY AND IMPLEMENTATION: Detailed documentation of this pipeline, along with a resource on latest FDA-approved CAR therapies is available on our website: https://fraunhofer-izi.github.io/Living-Drugs-Wiki/. The data underlying this article are available on GitHub at https://github.com/fraunhofer-izi/CERTOMICS. The code is also published on Zenodo at https://doi.org/10.5281/zenodo.18709693.
Multiomics↗
The cytotoxicity of mitomycin C and adriamycin in genetically engineered V79 cell lines and freshly isolated rat hepatocytes.
The objective of the present study was to investigate the cytotoxicity of Adriamycin (ADR) and mitomycin C (MMC) in tumor and non-tumor cells with respect to the role of cytochrome P450 (P450). Therefore, genetically engineered V79 Chinese hamster fibroblasts expressing only single enzymes of P450 were used. SD1 and XEM2 cells expressed rat P450IIB1 and P450IA1, respectively, whereas the V79 parental cells contained no detectable P450 levels. The cytotoxicity of ADR and MMC in the V79 cell system was compared with that in freshly isolated hepatocytes from phenobarbital (PB-hepatocytes)- and beta-naphthoflavone (beta NF-hepatocytes)-induced rats. Following 24 h of exposure to ADR equal cytotoxicity was observed in V79, SD1 and XEM2 cells. Addition of metyrapone (MP, an inhibitor of P450IIB1) and alpha-naphthoflavone (alpha NF, an inhibitor of P450IA1) had no effect on the ADR-induced cytotoxicity in SD1 and XEM2 cells, respectively. Likewise, MMC was equitoxic in V79 and SD1 cells. Co-incubation of SD1 cells with MP did not alter MMC-induced cytotoxicity. MMC, however, showed a decreased cytotoxicity in XEM2 cells when compared to the parental V79 cells. Unexpectedly, the cytotoxicity of MMC in XEM2 cells was increased by alpha NF to the same level as observed in the parental V79 cells. In contrast to V79- and V79-derived cells, in freshly isolated hepatocytes from PB or beta NF-induced rats, MMC was cytotoxic (measured as lactate dehydrogenase leakage) within 3 h of incubation. ADR, however, was only cytotoxic to the hepatocytes when intracellular glutathione was first depleted by diethylmaleate. The MMC- and ADR-induced cytotoxicity was found to be more pronounced in PB-hepatocytes than in beta NF-hepatocytes. Contrary to the findings in the V79-derived cells, MP afforded complete protection against both MMC- and ADR-induced cytotoxicity in PB-hepatocytes, whereas alpha NF only partially inhibited the cytotoxicity of MMC in beta NF-hepatocytes. In conclusion, we have demonstrated that PB-inducible P450s play a role in the cytotoxicity of both MMC and ADR in freshly isolated PB-hepatocytes but that P450IIB1 does not in genetically reconstituted SD1 cells. P450IA1, however, decreased the cytotoxicity of MMC in the XEM2 cells. The ADR-induced cytotoxicity, which was observed in XEM2 cells, was not mediated by P450IA1. The present study underscores the complexity in the comparison of ADR- and MMC-induced cytotoxicities in normal and tumor cells.
Animals↗
Enhanced in vivo monooxygenase activities of mammalian P450s in engineered yeast cells producing high levels of NADPH-P450 reductase and human cytochrome b5.
We have engineered yeast genomic DNA to construct a set of strains producing various relative amounts of yeast NADPH-P450 reductase (Yred) and human cytochrome b5 (Hb5). Expression of cDNAs encoding human P450 1A1, 1A2, 3A4, 19A and mouse P450 1A1 in the different oxido-reduction backgrounds thus constituted were achieved after strain transformation by plasmid-based P450-encoding expression cassettes. The results indicate that the level of Yred strongly affects all activities tested. In contrast, the amount of Hb5 affects activities in a manner that is dependent both on the P450 isoform considered and the Yred level. In a strain containing optimized amounts of Hb5 and Yred, human P450 3A4-specific testosterone-6 beta-hydroxylase activity can be enhanced as much as 73-fold in comparison with the activity observed in a wild-type strain. Bioconversion of sterols or xenobiotics was easily achieved in vivo using this new co-expression system.
Animals↗
Interleukin-2 production by tumor cells bypasses T helper function in the generation of an antitumor response.
A poorly immunogenic murine colon cancer was used to investigate mechanisms of antitumor immunity. Injection of tumor cells engineered by gene transfection to secrete IL-2 stimulated an MHC class I-restricted cytolytic T lymphocyte (CTL) response against the parental tumor. The tumor cells secreting IL-2 produced an antitumor response in vivo, even in the absence of CD4+ T cells. Animals immunized with the engineered cells were protected against subsequent challenge with the parental tumor cell line. Similar findings were demonstrated for other tumor types. Thus, provision of a helper lymphokine in a paracrine fashion induced a tumor-specific immune response involving activation of endogenous CTLs and other immune effector cells. These findings demonstrate that the failure of an effective antitumor immune response may be primarily due to a helper arm deficiency of the immune system rather than a paucity of tumor-specific cytotoxic effector cells. Furthermore, they outline a novel strategy for augmenting tumor immunity.
Animals↗
Cell Culture Engineering III. Conference proceedings. Florida, February 1-7, 1992.
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Animals↗
Mammalian PC-12 cell genetically engineered for human cytochrome P450 2E1 expression.
The stable expression of the human cytochrome CYP2E1 (P450 alcohol) was performed in the mammalian cell line PC-12. This cell line expressed cytochrome b5 (58 +/- 12 pmol/mg microsomal protein vs 528 +/- 80 pmol/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140 +/- 20 nmol.min-1.mg microsomal protein-1 vs 68 +/- 48 nmol.min-1.mg-1 in microsomal human liver). An expression plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co-transfected with the plasmid RSVneo into PC-12 cells. Clones were selected for resistance to the neomycin analog, G418, and then screened for expression of the CYP2E1 isozyme by testing for 6-hydroxylation of chlorzoxazone, a specific substrate for CYP2E1. Expression of CYP2E1 was confirmed in one clone, DB-7, by Western blot analysis and by measurement of monooxygenase activities which were not detectable in PC-12 cells. Chlorzoxazone 6-hydroxylation, n-butanol oxidation and dimethylnitrosamine N-demethylation were localized in microsomes (62, 60 and 63 pmol.min-1.mg microsomal protein-1, respectively) and were inhibited by carbon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzymes. Although the level of the enzyme activities was about a tenth of that measured in human liver microsomes, CYP2E1 expressed in DB-7 cells has catalytic competence similar to human liver CYP2E1. DB-7 cells metabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase. Construction of recombinant cell lines expressing CYP2E1 provides a useful tool for studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme.
1-Butanol↗
Transplantation of a polymer-encapsulated cell line genetically engineered to release NGF.
The delivery of nerve growth factor (NGF) to the lateral ventricle of a fimbria-fornix-lesioned rat prevents the lesion-induced reduction in choline acetyltransferase (ChAT) expression by medial septal cells. Although delivery has been achieved through neural grafting of genetically engineered cell lines which release NGF, transplanted cells have grown beyond the implantation site and formed tumors. The encapsulation of cells within a permselective polymer capsule prior to transplantation allows cell growth only within the capsule space, while allowing molecular exchange between the host tissue and enclosed cells. Rat fibroblasts from the parent cell line (R208F) or fibroblasts genetically modified to produce NGF (R208N.8) were loaded within a thermoplastic hollow fiber-based capsule. Only the capsules loaded with the genetically engineered cells released measurable amounts of NGF in culture. Adult rats received unilateral aspirative fimbria-fornix lesions, followed by intraventricular implantation of a R208F capsule (n = 6) or a R208N.8 capsule (n = 6). After 2 weeks, rats receiving encapsulated cells showed no undue reaction to the implants. With both cell types, the cells remained viable and confined to the capsule space. R208N.8 capsules released sufficient NGF to prevent the lesion-induced loss of septal ChAT expression, whereas R208F capsules did not. This study suggests that encapsulated genetically engineered cells can provide an efficient means for future applications involving delivery of neurotrophic factors.
Acetylcholinesterase↗
V79 Chinese hamster cells genetically engineered for stable expression of cytochromes P450.
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Animals↗
Chronic exposure to benzodiazepine receptor ligands uncouples the gamma-aminobutyric acid type A receptor in WSS-1 cells.
Chronic exposure to benzodiazepines can result in an "uncoupling" of gamma-aminobutyric acid (GABA) receptors and benzodiazepine receptors (BzR) both in primary neuronal cell cultures and in vivo. The effect of chronic exposure to BzR ligands was examined in an engineered cell line (WSS-1) stably expressing "type I" GABAA receptors. Chronic exposure to flurazepam produced a concentration- (EC50, approximately 1.1 microM after a 48-hr exposure) and time-dependent (t1/2, approximately 3 hr at 100 microM) reduction in the efficacy (Emax) of GABA to enhance [3H]flunitrazepam binding to BzR, a characteristic of uncoupling in native GABAA receptor isoforms. Uncoupling of GABAA receptors and BzR without concomitant changes in BzR density was also produced by chronic exposure to other, structurally diverse, BzR ligands, including Ro 15-1788 and methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, but was not manifested after exposure to the 5-hydroxytryptamine reuptake blocker fluoxetine. Chronic (12-48-hr) exposure to flurazepam did not remarkably alter levels of alpha 1 and gamma 2 mRNAs, which constitute GABAA receptors in this cell line. Based on these findings, it is hypothesized that uncoupling of GABAA receptors and BzR in this engineered cell line can proceed without the elaboration of additional novel subunits and could involve either post-translational modification of GABAA receptor proteins or changes in subunit stoichiometry.
Cell Line↗
Genetically engineered endothelial cells remain adherent and viable after stent deployment and exposure to flow in vitro.
Intravascular stents, currently in experimental human use for recurrent arterial stenosis, are plagued by subacute thrombosis. As a therapeutic approach to stent-related thrombosis, we and others have suggested coating stents with endothelial cells before implantation. In a previous study we demonstrated the feasibility of coating stents with endothelial cells that were genetically modified to secrete large amounts of human tissue plasminogen activator. In the present study we attempted both to develop a clinically applicable protocol for stent seeding and to test whether seeded cells would remain adherent to stents after exposure to pulsatile flow. Endothelial cells were harvested from the saphenous veins of sheep with survival of the donor animals. Harvested cells were transduced with a retroviral vector containing a marker gene and seeded onto catheter-mounted stents under sterile conditions. Scanning electron microscopy revealed complete coverage of the stent surfaces by seeded cells. Stents were expanded and exposed to pulsatile flow in vitro. Substantial cell retention was observed on the lateral stent surfaces by light microscopy and scanning electron microscopy; fewer cells were seen on the luminal and abluminal surfaces. Removal of seeded cells from flow-exposed stents by trypsin digestion resulted in the recovery of approximately 70% of the seeded cells. These cells were viable and healthy as judged by their ability to proliferate to confluence with the same kinetics as control (non-flow-exposed) cells. Autologous genetically modified endothelial cells can be seeded onto catheter-mounted stents in a sterile manner, and stent deployment under flow conditions results in substantial retention of viable cells.
Animals↗
Mammalian cell lines engineered to identify inhibitors of specific signal transduction pathways.
A variety of signal transduction pathways contribute to the regulation of transcription in mammalian cells. Several of these pathways ultimately rely upon the interaction of transcription factors with genetic sequences termed response elements in the promoter regions of some genes. The biochemical mechanisms that control the levels and state of activation of transcription factors are poorly understood. However, specific phosphorylation events mediated by protein kinase C, growth factor receptor-linked tyrosine kinases, and protein kinase A clearly participate in the regulation of these signal transduction pathways. To understand the relationship between activation and/or inhibition of these pathways and regulation of gene expression controlled by specific response elements, cell lines were prepared containing the TPA response element (TRE), serum response element (SRE), or cyclic AMP response element (CRE) fused to a gene encoding a secretable form of alkaline phosphatase (SEAP). These TRE-SEAP, SRE-SEAP, and CRE-SEAP cells exhibit dramatic increases in alkaline phosphatase (AP) activity following exposure to TPA, PDGF, or forskolin. Down regulation of protein kinase C or inhibition of tyrosine kinase activity blocked the stimulation of AP activity caused by TPA or PDGF. These cell lines can be used to characterize existing inhibitors, and to identify new agents that affect specific signal transduction pathways in mammalian cells.
1-Methyl-3-isobutylxanthine↗
Insect cell culture engineering. Serum-free media.
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Animals↗
Therapeutic gene delivery in human B-lymphoblastoid cells by engineered non-transforming infectious Epstein-Barr virus.
The B-lymphotrophic human herpes Epstein-Barr virus (EBV) is a 160-kilobase double-stranded DNA episomal virus carried in a persistent asymptomatic state by more than 90% of the worldwide adult population. We engineered a helper-dependent mini-EBV, with the minimal cis-EBV elements for episomal replication, viral amplification and packaging, for use as a gene delivery system. The therapeutic potential of this system was established by stably transducing B-lymphoblastoid cells from a Fanconi anaemia group C (FA-C) patient with a mini-EBV constitutively expressing the normal FACC cDNA and showing in vitro correction of the FA phenotype. In the absence of selective pressure, episomal expression persisted with a half-life of 30 days in actively growing transduced cells, indicating a retention rate of 98% expression per cell doubling. This work demonstrates the generation of an infectious non-transforming viral vector that can potentially deliver large therapeutic genes efficiently and selectively into human B cells.
B-Lymphocytes↗
A clinical evaluation of genetic stability.
Mutation in a genetically engineered cell line has not been shown to have been responsible for adverse clinical reactions in a currently licensed biotechnology product. There exists, however, the theoretical possibility that mutations in the production cell line could ultimately lead to patient exposure to aberrant proteins which could result in unanticipated or deleterious effects. The clinical trials of recombinant antihaemophilic factor, a large complex glycoprotein, which is administered chronically, offered a unique opportunity to explore this theoretical concern. In this retrospective analysis of patient data, evidence for unanticipated or adverse events which could possibly be attributed to mutations in the genetically engineered cell line was sought. Extensive analysis of a variety of patient data such as the efficacy, alterations in pharmacokinetic parameters, and inhibitor formation, gave no support for concern over host cell mutation. While this retrospective analysis cannot absolutely exclude the possibility of mutational events, those clinical data in combination with the product characterization information indicate that for this recombinant product, mutation is a highly improbable event.
Biotechnology↗
Artificial cell adhesive proteins engineered by grafting the Arg-Gly-Asp cell recognition signal: factors modulating the cell adhesive activity of the grafted signal.
An artificial cell adhesive protein could be engineered by grafting the RGDS tetrapeptide, the core sequence of the major cell adhesive site of fibronectin, to a truncated form of Staphylococcal protein A (tSPA) via cassette mutagenesis of the tSPA expression vector pRIT2T [T. Maeda et al. (1989) J. Biol. Chem. 264, 15165-15168]. We synthesized a panel of tSPA derivatives grafted with various RGDS-containing oligopeptides to address the problem of how the cell adhesive activity of the resulting tSPA derivatives was affected by the length and amino acid sequence of the grafted oligopeptides and by the sites on tSPA where the extra oligopeptides were inserted. The results showed that (i) the amino acid residues flanking the RGDS core sequence played a key role in modulating the cell adhesive activity of the grafted RGDS signal; (ii) at least two sites on tSPA, each corresponding to on e of the two HindIII sites of pRIT2T, were competent in sustaining the cell adhesive activity of the grafted signal; and (iii) the divalent tSPA containing the RGDS signal at both sites was more active than monovalent derivatives containing only one signal at either site. These results provide a strategic basis for engineering of artificial cell adhesive proteins by grafting the RGDS signal.
Amino Acid Sequence↗
Contribution of O6-alkylguanine and N-alkylpurines to the formation of sister chromatid exchanges, chromosomal aberrations, and gene mutations: new insights gained from studies of genetically engineered mammalian cell lines.
O6-methyl- and O6-ethylguanine are the major premutagenic and precarcinogenic lesions induced in DNA by monofunctional alkylating agents, albeit formed in minor amounts. The involvement of these lesions in SCE and aberration formation is less clear. We have analyzed the contribution of O6-alkylguanine to SCE and aberration formation, as well as its toxic and point mutation inducing effect in transgenic Chinese hamster ovary (CHO) cell lines that express variable amounts of human O6-methylguanine-DNA methyltransferase (MGMT). Cells that overexpress MGMT (or the bacterial Ada protein) gained resistance to the formation of alkylation-induced SCEs and aberrations, as compared to MGMT deficient cells. A correlation was apparent between the level of protection for SCEs and cell killing, indicating that both phenomena are interrelated. The protective effects were dependent on the level of MGMT expression, the agent used for alkylation, and cell cycle progression. Our data suggest that at least 2 kinds of lesions are responsible for SCE and aberration formation, namely, O6-alkylguanine and one or various N-alkylation products. The probability that O6-methylguanine is converted into cytogenetic effects has been estimated to be about 1:30 for SCEs, and 1:147,000 and 1:22,000 for chromosomal aberrations in the first and second post-treatment mitosis, respectively. The induction of SCEs and likely also of aberrations by O6-methylguanine requires two replication cycles and is supposed to involve the formation of secondary DNA lesions. Increased repair of 3-methyladenine and 7-methylguanine in CHO cells that overexpress the N-methylpurine-DNA glycosylase (MPG) after transfection with the human MPG-cDNA did not give rise to protection against methylation-induced SCEs and aberrations, probably because of incomplete excision repair. MPG overexpressing cells reacted even more sensitively to methylating agents, suggesting apurinic sites formed as a result of MPG action to be SCE and aberration-inducing lesions.
Alkylation↗