scholarly journals pIRES-CD4t, a Dicistronic Expression Vector for MACS- or FACS-Based Selection of Transfected Cells

BioTechniques ◽  
1999 ◽  
Vol 26 (4) ◽  
pp. 683-688 ◽  
Author(s):  
Peter Gaines ◽  
Don M. Wojchowski
Keyword(s):  
Expression Vector ◽  
Transfected Cells ◽  
Dicistronic Expression Vector ◽  
Selection Of

scholarly journals Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Hiroshi Sato ◽  
Hiroki Kato ◽  
Haruyoshi Yamaza ◽  
Keiji Masuda ◽  
Huong Thi Nguyen Nguyen ◽  
...  
Keyword(s):  
Normal Cell ◽  
Expression Vector ◽  
Gene Dosage ◽  
Genetic Disorders ◽  
Human Cells ◽  
Chromosome 21 ◽  
Disease Phenotypes ◽  
Simplex Virus ◽  
Selection Of ◽  
Trisomic Cell

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

scholarly journals COUP-TF acts as a competitive repressor for estrogen receptor-mediated activation of the mouse lactoferrin gene.

1993 ◽  
Vol 13 (3) ◽  
pp. 1836-1846 ◽  
Author(s):  
Y Liu ◽  
N Yang ◽  
C T Teng
Keyword(s):  
Estrogen Receptor ◽  
Expression Vector ◽  
Transfected Cells ◽  
Contact Sites ◽  
Estrogen Response ◽  
Upstream Promoter ◽  
Lactoferrin Gene ◽  
Thyroid Receptor ◽  
Estrogen Stimulation ◽  
Overlapping Region

We previously demonstrated that the estrogen response module (mERM) of the mouse lactoferrin gene, which contains an overlapping chicken ovalbumin upstream promoter transcription factor (COUP-TF)- and estrogen receptor-binding element, is responsible for estrogen induction. In this report we show that COUP-TF represses the mERM response to estrogen stimulation. Mutation and deletion of the COUP-TF-binding element or reduction of the endogenous COUP-TF increases mERM estrogen responsiveness. Likewise, overexpression of the COUP-TF expression vector blocked the estrogen-stimulated response of mERM in transfected cells. The molecular mechanism of this repression is due to the competition between COUP-TF and the estrogen receptor for binding at identical contact sites in the overlapping region of the mERM. Our results indicate that two members of the steroid-thyroid receptor superfamily work in concert to modulate lactoferrin gene expression.

pRIBOTEX expression vector: a pTEX derivative for a rapid selection of Trypanosoma cruzi transfectants

Gene ◽  
1997 ◽  
Vol 199 (1-2) ◽  
pp. 71-76 ◽  
Author(s):  
Santiago Martı́nez-Calvillo ◽  
Imelda López ◽  
Roberto Hernández
Keyword(s):  
Trypanosoma Cruzi ◽  
Expression Vector ◽  
Selection Of

scholarly journals Enhancement of Hepatitis C Virus RNA Replication by Cell Culture-Adaptive Mutations

2001 ◽  
Vol 75 (10) ◽  
pp. 4614-4624 ◽  
Author(s):  
Nicole Krieger ◽  
Volker Lohmann ◽  
Ralf Bartenschlager
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Colony Formation ◽  
Marker Gene ◽  
Rna Replication ◽  
Clear Correlation ◽  
Transfected Cells ◽  
Adaptive Mutations ◽  
Selection Of

ABSTRACT Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.

scholarly journals Magnetic Selection of Transiently Transfected Cells

BioTechniques ◽  
1996 ◽  
Vol 21 (5) ◽  
pp. 876-880 ◽  
Author(s):  
Stefan Schneider ◽  
Sandro Rusconi
Keyword(s):  
Transfected Cells ◽  
Selection Of

COUP-TF acts as a competitive repressor for estrogen receptor-mediated activation of the mouse lactoferrin gene

1993 ◽  
Vol 13 (3) ◽  
pp. 1836-1846
Author(s):  
Y Liu ◽  
N Yang ◽  
C T Teng
Keyword(s):  
Estrogen Receptor ◽  
Expression Vector ◽  
Transfected Cells ◽  
Contact Sites ◽  
Estrogen Response ◽  
Upstream Promoter ◽  
Lactoferrin Gene ◽  
Thyroid Receptor ◽  
Estrogen Stimulation ◽  
Overlapping Region

We previously demonstrated that the estrogen response module (mERM) of the mouse lactoferrin gene, which contains an overlapping chicken ovalbumin upstream promoter transcription factor (COUP-TF)- and estrogen receptor-binding element, is responsible for estrogen induction. In this report we show that COUP-TF represses the mERM response to estrogen stimulation. Mutation and deletion of the COUP-TF-binding element or reduction of the endogenous COUP-TF increases mERM estrogen responsiveness. Likewise, overexpression of the COUP-TF expression vector blocked the estrogen-stimulated response of mERM in transfected cells. The molecular mechanism of this repression is due to the competition between COUP-TF and the estrogen receptor for binding at identical contact sites in the overlapping region of the mERM. Our results indicate that two members of the steroid-thyroid receptor superfamily work in concert to modulate lactoferrin gene expression.

A Novel Mutation in the von Willebrand Factor Propeptide Causes Defective Regulated Storage in Platelets and Weibel-Palade Bodies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1007-1007
Author(s):  
Sandra L. Haberichter ◽  
Ulrich Budde ◽  
Elke Drewke ◽  
Tobias Obser ◽  
Cornelia Wermes ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Expression Vector ◽  
Intracellular Localization ◽  
Von Willebrand Disease ◽  
Relative Reduction ◽  
Homozygous Mutation ◽  
Transfected Cells ◽  
Storage Granules ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Two children from a Turkish consanguineous family suffering from significant mucocutaneous bleeding episodes were diagnosed with von Willebrand disease type 2A. The relative reduction of large plasma von Willebrand factor (VWF) multimers together with the absent triplet structure was consistent with type 2A (phenotype IIC) VWD. Surprisingly, platelet VWF was completely deficient of multimers beyond the VWF protomer, suggesting defective α-granular storage of larger multimers. The two patients were nearly unresponsive to DDAVP, consistent with a lack of regulated VWF release from endothelial cell Weibel-Palade bodies, suggesting defective storage in endothelial cells as well as in platelets. We identified the homozygous mutation in these patients, N528S, in the D2 domain of the VWF propeptide (VWFpp). To examine the effect of the N528S mutation on VWF regulated storage, a VWF expression vector containing the mutation was constructed and expressed in AtT-20 cells that contain an intact regulated storage pathway. Transfected cells were immunofluorescently labeled and the intracellular localization of VWFpp and VWF were examined by confocal microscopy. In cells expressing N528S-VWF, VWF was not stored in granules but was localized to the endoplasmic reticulum (ER). Our previous studies have shown that VWF granular storage is initiated by VWFpp: VWF is trafficked to storage granules through its non-covalent association with VWFpp. To determine if the loss of granular storage of N528S-VWF was due to defective VWFpp trafficking, an N528S-VWFpp expression vector was constructed and expressed in AtT-20 cells. The N528S-VWFpp was found to be colocalized with endogenous ACTH-containing storage granules, demonstrating normal granular trafficking of the variant VWFpp. The N528S-VWFpp was co-expressed with propeptide-deleted, mature VWF (Δpro) to examine regulated storage when the two proteins are expressed in trans. In the majority of transfected AtT-20 cells, N528S-VWFpp was trafficked to granules while the coexpressed Δpro appeared to be ER-localized, consistent with a loss of non-covalent association between VWFpp and VWF. In a subset of cells neither VWFpp nor VWF were stored in granules. In contrast, when N528S-VWF was expressed as a full-length construct (in cis), VWFpp granular storage was observed only in a subset of cells and the majority of transfected cells demonstrated ER-localization of both proteins, suggesting the loss of regulated storage of N528S-VWF is not due to defective VWFpp sorting, but rather an aberrant interaction of VWFpp with the mature VWF protein. Together these data indicate that the phenotype observed in patients with N528S variant VWF is result of defective multimerization, storage, and secretion due to an aberrant VWFpp/VWF interaction.

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