scholarly journals Simple and efficient custom transcription activator-like effector gene synthesis via twin primer assembly

BioTechniques ◽  
2021 ◽  
Author(s):  
Song Wang ◽  
Yan Yu ◽  
Yuhualei Pan ◽  
Huan Wang ◽  
Yushang Zhao ◽  
...  
Keyword(s):  
Low Cost ◽  
Gene Synthesis ◽  
Synthesis Methods ◽  
Kidney Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Transcription Activator ◽  
Human Embryonic Kidney Cells ◽  
Editing Efficiency ◽  
Double Stranded Dna

Transcription activator-like effector (TALE) nucleases (TALENs) efficiently recognize and cleave DNA in a sequence-dependent manner. However, current TALE custom synthesis methods are either complicated or expensive. Here we report a simple and low-cost method for TALE construct assembly. This method utilizes the denaturation/reannealing nature of double-stranded DNA to create a unique single-stranded DNA overhang for proper ordering of TALE monomers in an engineered multimer. We successfully synthesized two TALEN pairs targeting the endogenous TET1 locus in human embryonic kidney cells and demonstrated their editing efficiency. Our method provides an alternative simple, low-cost method for effective TALEN assembly, which may improve the application of TALE-based technology.

Ketamine Inhibits Monoamine Transporters Expressed in Human Embryonic Kidney 293 Cells 

1998 ◽  
Vol 88 (3) ◽  
pp. 768-774 ◽  
Author(s):  
Mitsuhiro Nishimura ◽  
Kohji Sato ◽  
Tomoya Okada ◽  
Ikuto Yoshiya ◽  
Patrick Schloss ◽  
...  
Keyword(s):  
Eukaryotic Expression ◽  
Psychotic Symptoms ◽  
Kidney Cells ◽  
Dependent Manner ◽  
Monoamine Transporters ◽  
Human Embryonic Kidney ◽  
General Anesthetic ◽  
Serotonin Transporters ◽  
Human Embryonic Kidney Cells ◽  
Dose Dependent

Background Ketamine has been characterized as having psychotomimetic and sympathomimetic effects. These symptoms have raised the possibility that ketamine affects monoaminergic neurotransmission. To elucidate the relation between ketamine and monoamine transporters, the authors constructed three cell lines that stably express the norepinephrine, dopamine, and serotonin transporters and investigated the effects of ketamine on these transporters. Methods Human embryonic kidney cells were transfected using the Chen-Okayama method with the human norepinephrine, rat dopamine, and rat serotonin transporter cDNA subcloned into the eukaryotic expression vector. Using cells stably expressing these transporters, the authors investigated the effects of ketamine on the uptake of these compounds and compared them with those of pentobarbital. Results Inhibition analysis showed that ketamine significantly inhibited the uptake of all three monoamine transporters in a dose-dependent manner. The Ki (inhibition constant) values of ketamine on the norepinephrine, dopamine, and serotonin transporters were 66.8 microM, 62.9 microM, and 162 microM, respectively. Pentobarbital, a typical general anesthetic agent with no psychotic symptoms, did not affect the uptake of monoamines, however. Further, neither the glycine transporter 1 nor the glutamate/aspartate transporter was affected by ketamine, indicating that ketamine preferentially inhibits monoamine transporters. Conclusions Ketamine inhibited monoamine transporters expressed in human embryonic kidney cells in a dose-dependent manner. This result suggests that the ketamine-induced inhibition of monoamine transporters might contribute to its psychotomimetic and sympathomimetic effects through potentiating monoaminergic neurotransmission.

Elevated carbon dioxide upregulates NBCn1 Na+/HCO3− cotransporter in human embryonic kidney cells

2013 ◽  
Vol 305 (12) ◽  
pp. F1765-F1774 ◽  
Author(s):  
Alejandro Orlowski ◽  
Lorena A. Vargas ◽  
Ernesto A. Aiello ◽  
Bernardo V. Álvarez
Keyword(s):  
Cell Types ◽  
Recovery Phase ◽  
Kidney Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Hek Cells ◽  
And Function ◽  
Acid Loading ◽  
Dose Dependent

The NBCn1 Na+/HCO3− cotransporter catalyzes the electroneutral movement of 1 Na+:1 HCO3− into kidney cells. We characterized the intracellular pH (pHi) regulation in human embryonic kidney cells (HEK) subjected to NH4Cl prepulse acid loading, and we examined the NBCn1 expression and function in HEK cells subjected to 24-h elevated Pco2 (10–15%). After acid loading, in the presence of HCO3−, ∼50% of the pHi recovery phase was blocked by the Na+/H+ exchanger inhibitors EIPA (10–50 μM) and amiloride (1 mM) and was fully cancelled by 30 μM EIPA under nominally HCO3−-free conditions. In addition, in the presence of HCO3−, pHi recovery after acid loading was completely blocked when Na+ was omitted in the buffer. pHi recovery after acidification in HEK cells was repeated in the presence of the NBC inhibitor S0859, and the pHi recovery was inhibited by S0859 in a dose-dependent manner ( Ki = 30 μM, full inhibition at 60 μM), which confirmed NBC Na+/HCO3− cotransporter activation. NBCn1 expression increased threefold after 24-h exposure of cultured HEK cells to 10% CO2 and sevenfold after exposure to 15% CO2, examined by immunoblots. Finally, exposure of HEK cells to high CO2 significantly increased the HCO3−-dependent recovery of pHi after acid loading. We conclude that HEK cells expressed the NBCn1 Na+/HCO3− cotransporter as the only HCO3−-dependent mechanism responsible for cellular alkaline loading. NBCn1, which expresses in different kidney cell types, was upregulated by 24-h high-Pco2 exposure of HEK cells, and this upregulation was accompanied by increased NBCn1-mediated HCO3− transport.

scholarly journals Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells

2021 ◽  
Vol 22 (1) ◽  
pp. 397
Author(s):  
Nasir Javaid ◽  
Thuong L. H. Pham ◽  
Sangdun Choi
Keyword(s):  
Protein Level ◽  
Reference Genes ◽  
Mrna Level ◽  
High Specificity ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Sox2 Expression

Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.

Fenton Reaction-Generated Advanced Oxidation Protein Products Induces Inflammation in Human Embryonic Kidney Cells

Inflammation ◽  
2016 ◽  
Vol 39 (4) ◽  
pp. 1285-1290 ◽  
Author(s):  
Guilherme Vargas Bochi ◽  
Vanessa Dorneles Torbitz ◽  
Roberto Christ Vianna Santos ◽  
Monica Cubillos-Rojas ◽  
José Luis Rosa López ◽  
...  
Keyword(s):  
Fenton Reaction ◽  
Advanced Oxidation ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Advanced Oxidation Protein Products ◽  
Human Embryonic Kidney Cells

scholarly journals Human embryonic kidney cells: stable transformation with an origin-defective simian virus 40 DNA and use as hosts for human papovavirus replication.

1984 ◽  
Vol 4 (2) ◽  
pp. 379-382 ◽  
Author(s):  
E O Major ◽  
P Matsumura
Keyword(s):  
Dna Replication ◽  
Sequence Data ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Defective Mutant ◽  
Dna Replication Origin ◽  
Human Papovavirus

An origin-defective mutant DNA of simian virus 40 immortalized human embryonic kidney cells, maintaining a T protein which could function for human papovavirus BK DNA replication but not for human papovavirus JC DNA replication. Neither BK virions nor capsid proteins were produced in these cells. This may indicate that the simian virus 40 T protein in human embryonic kidney cells is competent for maintaining transformation and initiating and completing DNA replication for BK but is not competent for switching to late gene functions. Furthermore, it appears that the JC DNA replication origin cannot efficiently use the simian virus 40 T protein for its DNA synthesis, as suggested by its DNA sequence data (R. Frisque, J. Virol. 46:170-176, 1983; T. Miyamura, H. Jikoya, E. Soeda, and K. Yoshiike, J. Virol. 45:73-79, 1983).

scholarly journals Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

2021 ◽  
Author(s):  
David A Hanna ◽  
Courtney M Moore ◽  
Liu Liu ◽  
Xiaojing Yuan ◽  
Angela S Fleischhacker ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Physiological Function ◽  
Hek293 Cells ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Heme Oxygenases ◽  
Heme Trafficking ◽  
Ferrous Heme

Heme oxygenases (HO) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms: inducible HO-1, which is up-regulated in response to various stressors, including excess heme, and constitutive HO-2. While much is known about the regulation and physiological function of HO-1, comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is largely dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or over-expressed HO-2, and various HO-2 mutant alleles, we found that endogenous heme is too limiting to support HO-2 catalyzed heme degradation. Rather, we discovered that a novel role for HO-2 is to bind and buffer labile heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor in control of heme bioavailability. When heme is in excess, HO-1 is induced and both HO-2 and HO-1 can provide protection from heme toxicity by enzymatically degrading it. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with the labile heme pool being oxidized, thereby providing new insights into heme trafficking and signaling.

A smart rhodamine–pyridine conjugate for bioimaging of thiocyanate in living cells

RSC Advances ◽  
2015 ◽  
Vol 5 (125) ◽  
pp. 103350-103357 ◽  
Author(s):  
Sandip Mandal ◽  
Animesh Sahana ◽  
Arnab Banerjee ◽  
Damir A. Safin ◽  
Maria G. Babashkina ◽  
...  
Keyword(s):  
Visible Light ◽  
Living Cells ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Eye Detection ◽  
Human Embryonic Kidney Cells ◽  
Naked Eye ◽  
Naked Eye Detection

A rhodamine–pyridine conjugate, REDA-2PC, can selectively monitor NCS− in human embryonic kidney cells 293. Visible light excitable probe allows fluorescence and naked eye detection of nanomolar NCS−.

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