scholarly journals Select Porcine Elongation Factor 1α Sequences Mediate Stable High-Level and Upregulated Expression of Heterologous Genes in Porcine Cells in Response to Primate Serum

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 1046
Author(s):  
Wu-Sheng Sun ◽  
Hyeon Yang ◽  
Jin Gu No ◽  
Haesun Lee ◽  
Nahyun Lee ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Human Leukocyte ◽  
Genetically Engineered ◽  
Regulatory Sequence ◽  
Elongation Factor 1Α ◽  
Cmv Promoter ◽  
Aortic Endothelial Cells ◽  
High Level ◽  
Genetic Profiles ◽  
Cag Promoter

Genetically engineered (GE) pigs with various combinations of genetic profiles have been developed using heterologous promoters. This study aimed to identify autologous promoters for high and ubiquitous expression of xenotransplantation relevant genes in GE pigs. A 1.4 kb upstream regulatory sequence of porcine elongation factor 1α (pEF1α) gene was selected and isolated for use as a promoter. Activity of the pEF1α promoter was subsequently compared with that of the cytomegalovirus (CMV) promoter, CMV enhancer/chicken β-actin (CAG) promoter, and human EF1α (hEF1α) promoter in different types of pig-derived cells. Comparative analysis of luciferase and mutant human leukocyte antigen class E-F2A-β-2 microglobulin (HLA-E) expression driven by pEF1α, CMV, CAG, and hEF1α promoters revealed the pEF1α promoter mediated comparable expression levels with those of the CAG promoter in porcine ear skin fibroblasts (PEFs) and porcine kidney-15 (PK-15) cells, but lower than those of the CAG promoter in porcine aortic endothelial cells (PAECs). The pEF1α promoter provided long-term stable HLA-E expression in PEFs, but the CAG promoter failed to sustain those levels of expression. For xenogeneic serum-induced cytotoxicity assays, the cells were cultured for several hours in growth medium supplemented with primate serum. Notably, the pEF1α promoter induced significant increases in luciferase and HLA-E expression in response to primate serum in PAECs compared with those driven by the CAG promoter, suggesting the pEF1α promoter could regulate temporal expression of heterologous genes under xenogeneic-cytotoxic conditions. These results suggest the pEF1α promoter may be valuable for development of GE pigs spatiotemporally and stably expressing immunomodulatory genes for xenotransplantation.

scholarly journals RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Wen Wang ◽  
Yan-long Jia ◽  
Yi-chun Li ◽  
Chang-qin Jing ◽  
Xiao Guo ◽  
...  
Keyword(s):  
Cho Cells ◽  
Transgene Expression ◽  
Recombinant Protein Expression ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Vector System ◽  
Gene Copy ◽  
Elongation Factor 1Α ◽  
Cmv Promoter ◽  
Expression Levels

Abstract In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Biochemical analysis of the interaction between elongation factor 1α and α/β-tubulins from a ciliate, Tetrahymena pyriformis

FEBS Letters ◽  
1999 ◽  
Vol 453 (1-2) ◽  
pp. 29-34 ◽  
Author(s):  
Masumi Nakazawa ◽  
David Moreira ◽  
Jacqueline Laurent ◽  
Hervé Le Guyader ◽  
Yasuo Fukami ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Elongation Factor ◽  
Tetrahymena Pyriformis ◽  
Elongation Factor 1Α

scholarly journals Molecular insights into the interaction of the ribosomal stalk protein with elongation factor 1α

2014 ◽  
Vol 42 (22) ◽  
pp. 14042-14052 ◽  
Author(s):  
Kosuke Ito ◽  
Takayoshi Honda ◽  
Takahiro Suzuki ◽  
Tomohiro Miyoshi ◽  
Ryo Murakami ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Elongation Factor 1Α ◽  
Ribosomal Stalk

G13A Substitution Affects the Biochemical and Physical Properties of the Elongation Factor 1α. A Reduced Intrinsic GTPase Activity Is Partially Restored by Kirromycin†

Biochemistry ◽  
2002 ◽  
Vol 41 (2) ◽  
pp. 628-633 ◽  
Author(s):  
Mariorosario Masullo ◽  
Piergiuseppe Cantiello ◽  
Barbara de Paola ◽  
Francesca Catanzano ◽  
Paolo Arcari ◽  
...  
Keyword(s):  
Physical Properties ◽  
Elongation Factor ◽  
Gtpase Activity ◽  
Elongation Factor 1Α ◽  
Intrinsic Gtpase Activity

Key aspects of the primary sex determination mechanism are conserved across the genus Drosophila

Development ◽  
1998 ◽  
Vol 125 (16) ◽  
pp. 3259-3268 ◽  
Author(s):  
J.W. Erickson ◽  
T.W. Cline
Keyword(s):  
Sex Determination ◽  
Rapid Onset ◽  
Amino Acid Identity ◽  
Bzip Transcription Factor ◽  
Regulatory Sequence ◽  
Tightly Coupled ◽  
Pole Cells ◽  
High Level ◽  
Key Aspects ◽  
Determination Mechanism

In D. melanogaster, a set of ‘X:A numerator genes’, which includes sisterlessA (sisA), determines sex by controlling the transcription of Sex-lethal (Sxl). We characterized sisA from D. pseudoobscura and D. virilis and studied the timing of sisA and Sxl expression with single cell-cycle resolution in D. virilis, both to guide structure-function studies of sisA and to help understand sex determination evolution. We found that D. virilis sisA shares 58% amino acid identity with its melanogaster ortholog. The identities confirm sisA as an atypical bZIP transcription factor. Although virilis sisA can substitute for melanogaster sisA, the protein is not fully functional in a heterologous context. The putative sisA regulatory sequence CAGGTAG is a potential ‘numerator box,’ since it is shared with the other strong X:A numerator gene, sisB, and its target, SxlPe. Temporal and spatial features of sisA and SxlPe expression are strikingly conserved, including rapid onset and cessation of transcription in somatic nuclei, early cessation of sisA transcription in budding pole cells and persistent high-level sisA expression in yolk nuclei. Expression of sisA and Sxl is as tightly coupled in virilis as it is in melanogaster. Taken together, these data indicate that the same primary sex determination mechanism exists throughout the genus Drosophila.

scholarly journals Database establishment for the secondary fungal DNA barcodetranslational elongation factor 1α(TEF1α)

Genome ◽  
2019 ◽  
Vol 62 (3) ◽  
pp. 160-169 ◽  
Author(s):  
Wieland Meyer ◽  
Laszlo Irinyi ◽  
Minh Thuy Vi Hoang ◽  
Vincent Robert ◽  
Dea Garcia-Hermoso ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Fungal Infections ◽  
Dna Barcode ◽  
Elongation Factor ◽  
Its Region ◽  
Reference Sequence ◽  
Diagnostic Tools ◽  
Reference Database ◽  
Elongation Factor 1Α ◽  
Fungal Dna

With new or emerging fungal infections, human and animal fungal pathogens are a growing threat worldwide. Current diagnostic tools are slow, non-specific at the species and subspecies levels, and require specific morphological expertise to accurately identify pathogens from pure cultures. DNA barcodes are easily amplified, universal, short species-specific DNA sequences, which enable rapid identification by comparison with a well-curated reference sequence collection. The primary fungal DNA barcode, ITS region, was introduced in 2012 and is now routinely used in diagnostic laboratories. However, the ITS region only accurately identifies around 75% of all medically relevant fungal species, which has prompted the development of a secondary barcode to increase the resolution power and suitability of DNA barcoding for fungal disease diagnostics. The translational elongation factor 1α (TEF1α) was selected in 2015 as a secondary fungal DNA barcode, but it has not been implemented into practice, due to the absence of a reference database. Here, we have established a quality-controlled reference database for the secondary barcode that together with the ISHAM-ITS database, forms the ISHAM barcode database, available online at http://its.mycologylab.org/ . We encourage the mycology community for active contributions.

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