scholarly journals Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

2017 ◽  
Author(s):  
Roman J. Szczesny ◽  
Katarzyna Kowalska ◽  
Kamila Klosowska-Kosicka ◽  
Aleksander Chlebowski ◽  
Ewelina P. Owczarek ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Efficiency ◽  
Protein Localization ◽  
Interaction Network ◽  
Bimolecular Fluorescence Complementation ◽  
Stable Cell Line ◽  
Dna Cloning ◽  
Stable Cell Lines ◽  
Mammalian Cell Lines ◽  
Recombination System

ABSTRACTDeciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. It may also be time-consuming if a homozygote is needed. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternative. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and creation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. The collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the utility of commonly used inducers of tetracycline-responsive promoters. We determined the protein interaction network of the exoribonuclease XRN2 and examined the role of the protein in transcription termination by RNAseq analysis of cells devoid of its ribonucleolytic activity. In total we created more than 500 DNA constructs which proves high efficiency of our strategy.

Effects of Succinic Acid Supplementation on Stable Cell Line Growth, Aggregation, and IgG and IgA Production

2020 ◽  
Vol 21 (10) ◽  
pp. 990-996
Author(s):  
Victoria Argentova ◽  
Teimur Aliev ◽  
Dmitry Dolgikh ◽  
Mikhail Kirpichnikov
Keyword(s):  
Cell Culture ◽  
Succinic Acid ◽  
Cell Lines ◽  
Recombinant Antibody ◽  
Lactate Production ◽  
Culture Method ◽  
Cell Aggregation ◽  
Stable Cell Line ◽  
Acid Supplementation ◽  
Stable Cell Lines

Background: Immunoglobulin (Ig) G is the most commonly used therapeutic antibodies. Recently, the interest in IgA antibodies to treat respiratory infectious diseases has been increasing. The reason for the inefficient use of IgA is recombinant antibody aggregation in cell culture, affecting the longevity and productivity of cell lines. Lactate is an important metabolite that affects the cultivation of stable cell lines producing monoclonal antibodies. Methods: In the present study, we investigated whether different combinations of succinic acid and micro-additives affect lactate production, which correlates with productivity. The effect of succinic acid substitution on productivity of cells producing IgG/IgA was analyzed using the static culture method in a six-well plate. Lactate was measured in supernatant of cell culture indirectly by using the activity of Lactate Dehydrogenase (LDH).A low lactate level was observed in cultivation medium supplemented with succinic acid or asparagine combined with some inorganic salts. Result: The results also demonstrated the effect of component supplementation on homogeneity, longevity, and productivity of cell culture. Supplementation of succinic acid eliminated cell aggregation and improved homogeneity of stable cell lines producing IgG and, especially, IgA. Conclusion: Overall, succinic acid supplementation to the culture medium has potential biotechnological applications in the production IgG and IgA.

scholarly journals Use of the Site-Specific Retargeting Jump-In Platform Cell Line to Support Biologic Drug Discovery

2014 ◽  
Vol 20 (4) ◽  
pp. 528-535 ◽  
Author(s):  
Robin Butler ◽  
David Hornigold ◽  
Ling Huang ◽  
Catherine Huntington ◽  
Tim London ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Cell Line ◽  
Cell Lines ◽  
Stable Cell Line ◽  
Therapeutic Antibodies ◽  
Site Specific ◽  
Mammalian Cell Lines ◽  
Cell To Cell Variability ◽  
High Degree

Biologics represent a fast-growing class of therapeutics in the pharmaceutical sector. Discovery of therapeutic antibodies and characterization of peptides can necessitate high expression of the target gene requiring the generation of clonal stably transfected cell lines. Traditional challenges of stable cell line transfection include gene silencing and cell-to-cell variability. Our inability to control these can present challenges in lead isolation. Recent progress in site-specific targeting of transgene to specific genomic loci has transformed the ability to generate stably transfected mammalian cell lines. In this article, we describe how the use of the Jump-In platform (Life Technologies, Carlsbad, CA) has been applied to drug discovery projects. It can easily and rapidly generate homogeneous high-expressing cell pools with a high degree of reproducibility. Their use in cell-based screening to identify specific binders, identify binding to relevant species variants, or detect functionally relevant therapeutic antibodies is central in driving drug discovery.

High Level Expression of Active Human Prothrombin in a Vaccina Virus Expression System

1992 ◽  
Vol 68 (02) ◽  
pp. 119-124 ◽  
Author(s):  
F G Falkner ◽  
P L Turecek ◽  
R T A MacGillivray ◽  
W Bodemer ◽  
F Scheiflinger ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cell Lines ◽  
Expression System ◽  
Human Cell Line ◽  
Cell System ◽  
Time Saving ◽  
Expression Levels ◽  
Mammalian Cell Lines ◽  
Cell Line Hela ◽  
Virus Expression

SummaryWe have worked out an efficient and time saving procedure for the expression of recombinant human prothrombin. The glycoprotein was expressed in the vaccinia virus expression system in several mammalian cell lines. The kidney cell lines Vero and BHK and the human cell line Hela were found to efficiently secrete prothrombin. Expression levels of 3–4 µg of factor II per 106 cells per day corresponding to 18–23 mU per 106 cells per day were achieved. Since the expression levels obtained with the vaccinia virus/Vero cell system were comparable to those obtained in amplified transformed CHO cells it provides an alternative system for the efficient expression of human prothrombin and may allow to further elucidate structure-function relationships of (pro)thrombin and its various effectors.

scholarly journals Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Cultured Cells ◽  
Hek293 Cells ◽  
Reaction Products ◽  
Enzyme Form ◽  
X Ray Crystallography ◽  
Mammalian Cell Lines ◽  
Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

scholarly journals Poly-L-ornithine-mediated transformation of mammalian cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2286-2293 ◽  
Author(s):  
V C Bond ◽  
B Wold
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
High Efficiency ◽  
Protein Product ◽  
Marker Genes ◽  
Recipient Mouse ◽  
L Cells ◽  
Dna And Rna ◽  
Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

RAS p21 and other Gn proteins are detected in mammalian cell lines by [γ-35S]GTPγS binding

1989 ◽  
Vol 159 (3) ◽  
pp. 1269-1274 ◽  
Author(s):  
J.G. Comerford ◽  
J.R. Gibson ◽  
A.P. Dawson ◽  
I. Gibson
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Mammalian Cell Lines ◽  
Gtpγs Binding ◽  
Ras P21

scholarly journals Novel B-cell precursors blocked at the stage of DJH recombination.

1988 ◽  
Vol 8 (12) ◽  
pp. 5216-5223 ◽  
Author(s):  
L Ramakrishnan ◽  
N Rosenberg
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Leukemia Virus ◽  
Variable Region ◽  
Immunoglobulin Gene ◽  
Recombination System ◽  
Rearrangement Process ◽  
B Cell Precursors ◽  
Enzymatic Machinery

Abelson murine leukemia virus-transformed cells have provided the principal model for study of the early events in immunoglobulin gene rearrangements. In this communication, we describe a new type of Abelson virus-transformed pre-B-cell line that is arrested at the DJH stage of the recombination process. These cells differ from other pre-B transformants with respect to two properties associated with the immunoglobulin rearrangement process. First, in contrast to cell lines undergoing VH-to-DJH joining in vitro, none of these cell lines contained detectable levels of RNAs transcribed from their unrearranged VH genes. Second, only some of the cell lines recombined exogenous heptamer-nonamer sequences, indicating that many of them have lost at least a portion of the enzymatic machinery that mediates recombination. The correlation between the absence of unrearranged VH RNAs and the inability to rearrange endogenous immunoglobulin gene segments suggests that VH gene transcription is required both to maintain an active recombination system and for the final step in variable-region formation.

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