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Correction: DAPE cloning with modified primers for producing designated lengths of 3' single-stranded ends in PCR products
[This corrects the article DOI: 10.1371/journal.pone.0318015.].
Journal Article
P05.02 Characterizing bispecific T-cell engagers with engineered CD3 effector and tailored target cells
BackgroundBi- and multi-specific modalities, with their numerous formats, are advancing cancer immunotherapy, particularly in hematologic malignancies. Created from components like immunoglobulin half molecules, Fab fragments, or single chain Fvs, they can recruit and activate immune cells or interfere with receptor signalling. Hundreds of such antibodies are in clinical development, demonstrating various modes of action. Blinatumomab, a Bispecific T-cell Engager (BiTE) molecule, targets CD3 and CD19 for T-cell mediated elimination of B-cell malignancies. CD19 is an attractive target because early B-cell malignancies arise from CD20-negative Pro-B and Pre-B cells, unlike Rituximab, which targets CD20. Consequently, blinatumomab was approved for aggressive B-cell precursor acute lymphoblastic leukemias (ALLs), often affecting children. This success has spurred development of other bispecific molecules for hematologic malignancies, with over two-thirds targeting CD19 and CD20 and a majority targeting CD3 on the effector side.MethodsWe engineered and cloned a reporter cell line based on the Jurkat human T-cell line, by stably expressing CD3 and a CD3-signaling responsive, NFAT-driven Firefly Luciferase reporter gene. Upon selection of the best performing clone a Renilla Luciferase expression construct under the control of a constitutively active promoter was added, allowing to assess potential toxic effect and normalization of the assay results. Based on the final clone, cell production was optimized to provide the basis for an unlimited source of frozen, ready-to-use cells.ResultsUsing the BiTE Blinatumomab, we showcase that our above-described CD3-responsive effector cells reply in a specific and dose responsive manner to the presence of Blinatumomab and CD19 positive target cells. In addition, we demonstrate the assay performance and robustness in different application settings.ConclusionAnalytical methods for BiTEs based on primary cells have limitations which can be overcome with our iLite bioassay platform allowing to assess CD3-dependent MoAs in an easy and reliable way. Despite not shown here, the cells were proven to work also in the context of a variety of BiTEs directed against a multitude of different antigen targets. Beyond that, this cell line can be a starting point for further cell line engineering to reflect additional MoAs on top of CD3-based T-cell engagement of more complex modalities. The user-friendly, frozen, Assay Ready Cell format allows a maximum of flexibility while providing highest assay consistency in the contexts of drug screening and characterization as well as in drug potency and immunogenicity assessment.All authors are employees of Svar Life Science
Journal Article
The ABCs of gene cloning
Clear and concise, this easy-to-use book offers an introductory course on the language of gene cloning, covering microbial, plant, and mammalian systems. It presents the nuts and bolts of gene cloning in a well-organized and accessible manner. Part I of this book outlines the essentials of biology and genetics relevant to the concept of gene cloning. Part II describes common techniques and approaches of gene cloning, ranging from the basic mechanics of DNA manipulation, vector systems, process transformation, to gene analysis. Part III & IV present application technologies of major impact in agriculture, biomedicine, and related areas. The ABCs of Gene Cloning, Third Edition contains updates including a tutorial chapter on gene-vector construction, methodologies on exome sequencing in finding disease genes, revised topics on gene therapy and whole genome sequencing, new developments for gene targeting and genome editing, as well as the current state of next generation sequencing. With more than 140 illustrations, this new edition provides an invaluable text for students and anyone who have interest in gaining proficiency in reading and speaking the language of gene cloning.
Book
Accumulation of Alkaloids in Different Tall Fescue KY31 Clones Harboring the Common Toxic IEpichloë coenophiala/I Endophyte under Field Conditions
Tall fescue (Lolium arundinaceum) is a highly adaptable forage, pasture and turf grass that is grown on over 14 M ha in the eastern half of the United States and in other temperate regions of the world. A significant factor in adaptability, productivity and stand persistence is in part due to the presence of an intercellular, seed-transmissible, endophytic fungus, Epichloë coenophiala. Epichloë endophytes have been shown to produce a number of alkaloid compounds only in planta, some that are beneficial in repelling insects, while others are toxic to animals. The goal of this work was to monitor the level of the ergot and loline (classified as pyrrolizidine) alkaloid accumulation in individual plants to determine the plant genotype contribution to alkaloid concentrations. The experimental design consisted of sixteen tall fescue KY31 clones in a space-planted, replicated trial over three years. Our results demonstrated that while changes in the alkaloid concentrations for each plant/endophyte genotype were observed over the three years, the overall alkaloid levels remained relatively constant when compared to other plant/endophyte genotypes combinations in the field. Additionally, overall levels of the ergot and loline alkaloid accumulation did not vary in the same way over the three years. Since the E. coenophiala endophyte genotype was the same across all clones, our results indicate that it is the plant genotype that is responsible for determining alkaloid levels in each plant, and suggest that the signal(s) from the plant to the endophyte may not be the same for ergot and loline alkaloid production.
Journal Article
De-extinction : the science of bringing lost species back to life
In the twenty-first century, because of climate change and other human activities, many animal species have become extinct, and many others are at risk of extinction. Once they are gone, we cannot bring them back or can we?
Book
Correction: A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications
(B) Schematic of lentiviral destination vectors pEpic and pEpic_Lite. attR3 and 4 sites flanking the ccdB/CmR selection cassette are positioned in an anti-sense orientation to viral RNA expression driven by a Rous sarcoma virus (RSV) promoter. pEpic_Lite lacks puromycin resistance (PuroR).LTR = long terminal repeat; RRE = Rev response element; cPPT = central polypurine tract; ccdB = E. coli ccdB toxin; CmR = chloramphenicol resistance; mPGK = mouse phosphoglycerate kinase promoter; WPRE = woodchuck hepatitis virus posttranslational regulatory element. https://doi.org/10.1371/journal.pone.0176543.g001 The correct plasmids and their sequences have been deposited with Addgene (www.addgene.org), with the following catalog numbers: pEpic #84372; pEpic_Lite #84373.
Journal Article
Cloning and genetic engineering
This enlightening volume offers arguments for both sides of the cloning and genetic engineering debate.
Book
EasyClone: method for iterative chromosomal integration of multiple genes Saccharomyces cerevisiae
Abstract
Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.
EasyClone genetical toolbox allows faster development of yeast strains for biotechnological applications.
Journal Article