scholarly journals Control of Multigene Expression Stoichiometry in Mammalian Cells Using Synthetic Promoters

2021 ◽  
Author(s):  
Yash D. Patel ◽  
Adam J. Brown ◽  
Jie Zhu ◽  
Guglielmo Rosignoli ◽  
Suzanne J. Gibson ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Synthetic Promoters ◽  
Multigene Expression

scholarly journals Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures

2021 ◽  
Author(s):  
Ileana Tossolini ◽  
Agustina Gugliotta ◽  
Fernando Lopez Diaz ◽  
Ricardo Kratje ◽  
Claudio Prieto
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
High Strength ◽  
High Rate ◽  
High Expression ◽  
Expression Vectors ◽  
Cmv Promoter ◽  
Promising Alternative ◽  
Expression Levels ◽  
Synthetic Promoters

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, endogenous promoters were identified based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.

scholarly journals A plasmid-based multigene expression system for mammalian cells

2010 ◽  
Vol 1 (1) ◽  
Author(s):  
Andrijana Kriz ◽  
Katharina Schmid ◽  
Nadia Baumgartner ◽  
Urs Ziegler ◽  
Imre Berger ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
Multigene Expression

DNA sequence requirements for transcriptional initiator activity in mammalian cells

1994 ◽  
Vol 14 (1) ◽  
pp. 116-127
Author(s):  
R Javahery ◽  
A Khachi ◽  
K Lo ◽  
B Zenzie-Gregory ◽  
S T Smale
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Tata Box ◽  
Start Site ◽  
Sequence Element ◽  
Synthetic Promoters ◽  
Sequence Requirements

A transcriptional initiator (Inr) for mammalian RNA polymerase II can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for (i) determining the start site location in a promoter that lacks a TATA box and (ii) enhancing the strength of a promoter that contains a TATA box. We have prepared synthetic promoters containing random nucleotides downstream of Sp1 binding sites to determine the range of DNA sequences that convey Inr activity. Numerous sequences behaved as functional Inrs in an in vitro transcription assay, but the Inr activities varied dramatically. An examination of the functional elements revealed loose but consistent sequence requirements, with the approximate consensus sequence Py Py A+1 N T/A Py Py. Most importantly, almost every functional Inr that has been described fits into the consensus sequence that we have defined. Although several proteins have been reported to bind to specific Inrs, manipulation of those elements failed to correlate protein binding with Inr activity. The simplest model to explain these results is that all or most Inrs are recognized by a universal binding protein, similar to the functional recognition of all TATA sequences by the same TATA-binding protein. The previously reported proteins that bind near specific Inr elements may augment the strength of an Inr or may impart transcriptional regulation through an Inr.

scholarly journals DNA sequence requirements for transcriptional initiator activity in mammalian cells.

1994 ◽  
Vol 14 (1) ◽  
pp. 116-127 ◽  
Author(s):  
R Javahery ◽  
A Khachi ◽  
K Lo ◽  
B Zenzie-Gregory ◽  
S T Smale
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Tata Box ◽  
Start Site ◽  
Sequence Element ◽  
Synthetic Promoters ◽  
Sequence Requirements

A transcriptional initiator (Inr) for mammalian RNA polymerase II can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for (i) determining the start site location in a promoter that lacks a TATA box and (ii) enhancing the strength of a promoter that contains a TATA box. We have prepared synthetic promoters containing random nucleotides downstream of Sp1 binding sites to determine the range of DNA sequences that convey Inr activity. Numerous sequences behaved as functional Inrs in an in vitro transcription assay, but the Inr activities varied dramatically. An examination of the functional elements revealed loose but consistent sequence requirements, with the approximate consensus sequence Py Py A+1 N T/A Py Py. Most importantly, almost every functional Inr that has been described fits into the consensus sequence that we have defined. Although several proteins have been reported to bind to specific Inrs, manipulation of those elements failed to correlate protein binding with Inr activity. The simplest model to explain these results is that all or most Inrs are recognized by a universal binding protein, similar to the functional recognition of all TATA sequences by the same TATA-binding protein. The previously reported proteins that bind near specific Inr elements may augment the strength of an Inr or may impart transcriptional regulation through an Inr.

Ultrastructural Alterations in Hela Cells Induced by Spider Venom

1967 ◽  
Vol 25 ◽  
pp. 20-21
Author(s):  
Dale E. McClendon ◽  
Paul N. Morgan ◽  
Bernard L. Soloff
Keyword(s):  
Growth Medium ◽  
Mammalian Cells ◽  
Carcinoma Cells ◽  
Venom Protein ◽  
Sterile Saline ◽  
Carbon Coated ◽  
Available Information ◽  
Loxosceles Reclusa ◽  
Essential Growth ◽  
Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Dissection of the molecular mechanisms of protein targeting to the peroxisome using immunofluorescence and immunocryoelectron microscopies

1990 ◽  
Vol 48 (3) ◽  
pp. 44-45
Author(s):  
G-A. Keller ◽  
S. J. Gould ◽  
S. Subramani ◽  
S. Krisans
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Protein Targeting ◽  
Firefly Luciferase ◽  
Peroxisomal Enzyme ◽  
Transfected Cells ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Series Of Experiments ◽  
Peroxisomal Targeting Signal

Subcellular compartments within eukaryotic cells must each be supplied with unique sets of proteins that must be directed to, and translocated across one or more membranes of the target organelles. This transport is mediated by cis- acting targeting signals present within the imported proteins. The following is a chronological account of a series of experiments designed and carried out in an effort to understand how proteins are targeted to the peroxisomal compartment.-We demonstrated by immunocryoelectron microscopy that the enzyme luciferase is a peroxisomal enzyme in the firefly lantern. -We expressed the cDNA encoding firefly luciferase in mammalian cells and demonstrated by immunofluorescence that the enzyme was transported into the peroxisomes of the transfected cells. -Using deletions, linker insertions, and gene fusion to identify regions of luciferase involved in its transport to the peroxisomes, we demonstrated that luciferase contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acid.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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