Expression of recombinant protein in cho and hela cells and its follow-up using EGF reporter gene

2006 ◽  
pp. 55-59
Author(s):  
V. Hendrick ◽  
D. Ribeiro Sousa ◽  
A. R. dos Santos Pedregal ◽  
C. Bassens ◽  
P. Rigaux ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Reporter Gene ◽  
Hela Cells

scholarly journals The AI196 antibody recognizes a SPOT-tagged recombinant protein by immunofluorescence

2020 ◽  
Vol 3 (2) ◽  
pp. e136
Author(s):  
Wanessa C Lima ◽  
Pierre Cosson
Keyword(s):  
Recombinant Protein ◽  
Hela Cells ◽  
Spot Tag

The AI196 antibody against the SPOT tag recognizes a SPOT-tagged human TAC protein by immunofluorescence in paraformaldehyde-fixed HeLa cells.

scholarly journals AE391 and AF291 antibodies recognize an HA-tagged recombinant protein by immunofluorescence

2020 ◽  
Vol 3 (2) ◽  
pp. e133
Author(s):  
Wanessa C Lima ◽  
Pierre Cosson
Keyword(s):  
Recombinant Protein ◽  
Hela Cells

AE391 and AF291 antibodies against the HA tag recognize an HA-tagged human TAC protein by immunofluorescence in paraformaldehyde-fixed HeLa cells.

scholarly journals The AI215 antibody recognizes an EPEA-tagged recombinant protein by immunofluorescence

2020 ◽  
Vol 3 (2) ◽  
pp. e130
Author(s):  
Wanessa C Lima ◽  
Pierre Cosson
Keyword(s):  
Recombinant Protein ◽  
Hela Cells

The AI215 antibody against the EPEA tag recognizes an EPEA-tagged human TAC protein by immunofluorescence in paraformaldehyde-fixed HeLa cells.

scholarly journals A silencer element for the lipoprotein lipase gene promoter and cognate double- and single-stranded DNA-binding proteins.

1995 ◽  
Vol 15 (1) ◽  
pp. 517-523 ◽  
Author(s):  
Y Tanuma ◽  
H Nakabayashi ◽  
M Esumi ◽  
H Endo
Keyword(s):  
Dna Binding ◽  
Lipoprotein Lipase ◽  
Reporter Gene ◽  
Hela Cells ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Binding Factor ◽  
Lpl Gene ◽  
Silencer Element

Transfection experiments with constructs containing various 5'-deleted fragments of the human lipoprotein lipase (LPL) promoter and the chloramphenicol acetyltransferase reporter gene revealed an LPL silencer element (LSE) in the region of nucleotides -225 to -81 of the LPL gene that functioned in Chinese hamster ovary (CHO) and HeLa cells. Gel retardation competition analysis showed the presence of a nuclear factor(s) capable of binding to the sequence of nucleotides -169 to -152 of LSE (LSE-6) in a single-stranded (opposite-strand) and double-stranded specific fashion, the binding affinity being almost the same in the two binding forms. Site-directed mutagenesis indicated that almost the entire sequence of LSE-6 was necessary to form the complexes and also critical for silencing activity in CHO cells. The amounts of this binding factor(s) in CHO and HeLa cells were closely associated with transcriptional silencing activity. Photochemical cross-linking experiments indicated that the single- and double-stranded elements recognized the same binding factor(s) with molecular masses of 54 to 63 kDa and 109 to 124 kDa. The 109- to 124-kDa DNA binding factor(s) was found to be a doublet of that of the 54- to 63-kDa factor by isoelectric focusing or by increasing the time of exposure to UV irradiation. When inserted upstream of another gene such as that of the simian virus 40 enhancer/promoter of pSV2CAT, the sequence of nucleotides -190 to -143 (LSE-1) also suppressed transcription of the reporter gene in CHO cells. These results strongly suggest that the LSE plays a role in regulation of LPL gene expression by suppressing its transcription.

scholarly journals Regulatory role of MEF2D in serum induction of the c-jun promoter.

1995 ◽  
Vol 15 (6) ◽  
pp. 2907-2915 ◽  
Author(s):  
T H Han ◽  
R Prywes
Keyword(s):  
Dna Binding ◽  
Reporter Gene ◽  
Hela Cells ◽  
Serum Response Factor ◽  
Common Mechanism ◽  
Response Factor ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Serum Response ◽  
Nih 3T3 Cells

Serum induction of c-jun expression in HeLa cells requires a MEF2 site at -59 in the c-jun promoter. MEF2 sites, found in many muscle-specific enhancers, are bound by a family of transcription factors, MEF2A through -D, which are related to serum response factor in their DNA binding domains. We have found that MEF2D is the predominant protein in HeLa cells that binds to the c-jun MEF2 site. Serum induction of a MEF2 reporter gene was not observed in a line of NIH 3T3 cells which contain low MEF2 site binding activity. Transfection of MEF2D into NIH 3T3 cells reconstituted serum induction, demonstrating that MEF2D is required for the serum response. Deletion analysis of MEF2D showed that its DNA binding domain, when fused to a heterologous transcriptional activation domain, was sufficient for serum induction of a MEF2 reporter gene. This is the domain homologous to that in the serum response factor which is required for serum induction of the c-fos serum response element, suggesting that serum regulation of c-fos and c-jun may share a common mechanism.

Comparing normal primary endocervical adenoepithelial cells to uninfected and influenza B virus infected human cervical adenocarcinoma HeLa cells

2006 ◽  
Vol 16 (6) ◽  
pp. 2032-2038 ◽  
Author(s):  
D. O. Sioutopoulou ◽  
E. T. Plakokefalos ◽  
G. M. Anifandis ◽  
L. D. Arvanitis ◽  
I. Venizelos ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Hela Cells ◽  
Staining Method ◽  
Respiratory Viruses ◽  
G1 Phase ◽  
Influenza B Virus ◽  
Influenza B ◽  
B Virus ◽  
Abnormal Cells

Human adenocarcinoma HeLa cells surviving infection with low (10−9units), medium (10−6units), and high (10−2units) influenza B titers were compared to their uninfected precursors and to normal endocervical adenoepithelial and metaplastic cells using Papanikolaou-staining method and immunocytochemistry. Normal primary endocervical and infected HeLa cells surviving infection shared similar morphologic, phenotypic, and divisional patterns that differed drastically from those of uninfected HeLa cells. The number of infected hosts surviving 6–7 days of viral exposure did not change during 3-week follow-up period, and their cyclin E levels suggested that they had been arrested to the G1 phase of the cell cycle by viral stress. Our findings suggest that in addition to apoptosis, nononcogenic viral stress activated the expression of endocervical metaplastic-like motifs in surviving hosts. A mechanism of cell response to nononcogenic viral stress was proposed to explain these findings. We conclude that nononcogenic respiratory viruses specifically target and eliminate abnormal cells ectopically overexpressing appropriate receptors and may complement current treatments of cervical cancer.

scholarly journals The mouse androgen receptor. Functional analysis of the protein and characterization of the gene

1991 ◽  
Vol 278 (1) ◽  
pp. 269-278 ◽  
Author(s):  
P W Faber ◽  
A King ◽  
H C J van Rooij ◽  
A O Brinkmann ◽  
N J de Both ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Reporter Gene ◽  
Hela Cells ◽  
Transcription Initiation ◽  
Promoter Region ◽  
Sequence Similarity ◽  
Dependent Manner ◽  
S1 Nuclease ◽  
Isolation And Characterization

Screening a mouse genomic DNA library with human androgen-receptor (hAR) cDNA probes resulted in the isolation and characterization of eight genomic fragments that contain the eight exons of the mouse androgen-receptor (mAR) gene. On the basis of similarity to the hAR gene, the nucleotide sequences of the protein-coding parts of the exons as well as the sequences of the intron/exon boundaries were determined. An open reading frame (ORF) of 2697 nucleotides, which can encode an 899-amino-acid protein, could be predicted. The structure of the mAR ORF was confirmed by sequence analysis of mAR cDNA fragments, which were obtained by PCR amplification of mouse testis cDNA, using mAR specific primers. A eukaryotic mAR expression vector was constructed and mAR was transiently expressed in COS-1 cells. The expressed protein was shown by Western blotting to be identical in size with the native mAR. Co-transfection of HeLa cells with the mAR expression plasmid and an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter-gene construct showed mAR to be able to trans-activate the androgen-responsive promoter in a ligand-dependent manner. Transcription-initiation sites of the mAR gene were identified by S1-nuclease protection experiments, and the functional activity of the promoter region was determined by transient expression of mAR promoter-CAT-reporter-gene constructs in HeLa cells. Structural analysis revealed the promoter of the mAR gene to be devoid of TATA/CCAAT elements. In addition, the promoter region is not remarkably (G + C)-rich. Potential promoter elements consist of a consensus Sp1 binding sequence and a homopurine stretch. The polyadenylation sites of mAR mRNA were identified by sequence similarity to the corresponding sites in the hAR mRNA.

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