scholarly journals Enhancement of Transgene Expression by Mild Hypothermia Is Promoter Dependent in HEK293 Cells

Life ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 901
Author(s):  
Min Ho Jang ◽  
Honggi Min ◽  
Jae Seong Lee
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Mild Hypothermia ◽  
Hek293 Cells ◽  
Egfp Expression ◽  
Sequencing Analysis ◽  
Cmv Promoter ◽  
Promoter Sequences ◽  
Hypothermic Response ◽  
Cmv Enhancer

Mild hypothermia has been widely used to enhance transgene expression and improve the cellular productivity of mammalian cells. This study investigated mild hypothermia-responsive exogenous promoters in human embryonic kidney 293 (HEK293) cells using site-specific integration of various promoter sequences, including CMV, EF1α, SV40, and TK promoters, into the well-known genomic safe harbor site, AAVS1. EGFP expression driven by the CMV promoter increased up to 1.5-fold at 32 °C versus 37 °C under stable expression, while others showed no hypothermic response. Integration of short CMV variants revealed that the CMV-enhancer region is responsible for the positive hypothermic response. CMV-enhancer-specific transcription factors (TFs) were then predicted through in silico analysis and RNA-sequencing analysis, resulting in the selection of one TF, NKX3-1. At 37 °C, overexpression of NKX3-1 in recombinant HEK293 cells expressing EGFP through the CMV promoter (CMV-EGFP) increased EGFP expression up to 1.6-fold, compared with that in CMV-EGFP, the expression level of which was comparable to that of CMV-EGFP at 32 °C. Taken together, this work demonstrates promoter-dependent hypothermia responses in HEK293 cells and emphasizes interactions between endogenous TFs and promoter sequences.

scholarly journals Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

2019 ◽  
Author(s):  
Binhui Zhao ◽  
Pankaj Chaturvedi ◽  
David L. Zimmerman ◽  
Andrew S. Belmont
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Large Fraction ◽  
Fine Tuning ◽  
Cmv Promoter ◽  
Chromosome Position ◽  
High Level ◽  
Genomic Regions ◽  
Expression In Mammalian Cells

ABSTRACTAchieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC containing the mouseMsh3-Dhfrlocus (DHFR BAC). Here we extend this “BAC TG-EMBED” approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01-5 fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes expressed on a single BAC. Second, we show small variability in both the expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making choice of BACs more flexible. Third, we describe an intriguing phenomenon in which BAC transgenes are maintained as episomes in a large fraction of stably selected clones. Finally, we demonstrate the utility of BAC TG-EMBED by simultaneously labeling three nuclear compartments in 94% of stable clones using a multi-reporter DHFR BAC, constructed with a combination of synthetic biology and BAC recombineering tools. Our extended BAC TG-EMBED method provides a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies.

Nuclear-Associated Plasmid, But Not Cell-Associated Plasmid, is Correlated With Transgene Expression in Cultured Mammalian Cells

2000 ◽  
Author(s):  
Molly B. James ◽  
Todd D. Giorgio
Keyword(s):  
Hela Cell ◽  
Hela Cells ◽  
Plasmid Dna ◽  
Mammalian Cells ◽  
Transgene Expression ◽  
Quantitative Method ◽  
Fluorescent Protein ◽  
Cmv Promoter ◽  
Green Fluorescent ◽  
Plasmid Delivery

Abstract Intracellular plasmid is rapidly incorporated into the nucleus of HeLa cells following cationic lipoplex transfection. CV1 cells are less effective in translocating plasmid to the nucleus and also express less transgene than HeLa cells. Cultured HeLa and CV1 cells and corresponding isolated nuclei were analyzed after transfection of a Cy3 labeled pGreenLantern plasmid (Cy3-pGL). Flow cytometry was used to measure both plasmid delivery and transgene expression from the plasmid encoding a CMV promoter driven green fluorescent protein. During transfection, HeLa cells rapidly incorporated the plasmid, reaching a maximum of 80% Cy3-pGL positive cells 8 hours post-transfection. The average Cy3-pGL positive HeLa cell contained approximately 2470 plasmid copies. 48% of the nuclei isolated from the transfected HeLa cells were positive for the plasmid marker after 8 hours. In contrast to HeLa cells, fewer CV1 cells and CV1 nuclei incorporated plasmid DNA with peak transfection occurring after 12 hours for 36% of the cells and after 8 hours for 12% of the nuclei. However, the average Cy3-pGL positive CV1 cell did not have a significantly different number of total cellular plasmid copies than the average positive HeLa cell. CV1 nuclei, however, had half as much plasmid as HeLa nuclei. HeLa cells are more efficient than CV1 cells at transporting plasmid from the cytoplasm to the nucleus. This study demonstrates the use of a novel quantitative method to study plasmid transport from the cytoplasm to nucleus and the effect on transgene expression.

An efficient system for tissue-specific overexpression of transgenes in podocytes in vivo

2005 ◽  
Vol 289 (2) ◽  
pp. F481-F488 ◽  
Author(s):  
Marcus J. Moeller ◽  
Abdulsalam Soofi ◽  
Silja Sanden ◽  
Jürgen Floege ◽  
Wilhelm Kriz ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Fluorescent Protein ◽  
Cost Effective ◽  
Egfp Expression ◽  
Cmv Promoter ◽  
Efficient System ◽  
Tissue Specific ◽  
Cre Recombination ◽  
Green Fluorescent

The utility of promoter fragments isolated from the 5′-flanking region of endogenous mammalian genes to drive transgene expression in vivo is often limited by low expression levels. In this study, a bigenic system was established that allows constitutive overexpression of transgenes in a tissue-specific fashion in transgenic mice in a time- and cost-effective fashion. A modified floxed expression vector was constructed [CMVflox-enhanced green fluorescent protein (eGFP)], in which a lacZ cassette (β-galactosidase) flanked by lox sites was placed between a CMV-promoter and the transgene of interest (eGFP). Before Cre recombination, expression of eGFP was effectively prevented by the interposed floxed lacZ cassette, whereas β-galactosidase was strongly expressed in transiently transfected cells. Transcription of the gene of interest (eGFP) could be irreversibly activated by cotransfection with Cre recombinase. Mice transgenic for CMVflox-eGFP were generated by pronuclear injection. A rapid assay was developed to identify transgenic founders with active transgene expression by measuring transgene activity (β-galactosidase) in tail biopsies. Transgene activity in tails correlated with transgene expression in most other tissues tested including podocytes within the kidney. To activate expression of the gene of interest in a tissue-specific fashion, founder mice were mated to the Cre mouse line 2.5P-Cre previously shown to mediate 100% Cre recombination exclusively in podocytes (Moeller MJ, Sanden SK, Soofi A, Wiggins RC, and Holzman LB. Genesis 35: 39–42, 2003). In doubly transgenic offspring, high-level eGFP expression resulting from Cre excision of the interposed lacZ cassette was detected in four of seven CMVflox-eGFP founder lines. This approach should also circumvent common limitations arising from lethality or transgene silencing as a consequence of transgene overexpression.

scholarly journals Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1391
Author(s):  
Andrey Kropotov ◽  
Veronika Kulikova ◽  
Kirill Nerinovski ◽  
Alexander Yakimov ◽  
Maria Svetlova ◽  
...  
Keyword(s):  
Nicotinic Acid ◽  
Mammalian Cells ◽  
Experimental Models ◽  
The Body ◽  
Nucleoside Transporters ◽  
Hek293 Cells ◽  
Nucleoside Transporter ◽  
Vitamin B3 ◽  
Animal Cells ◽  
Nicotinamide Riboside

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

scholarly journals Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1–9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Thu T. Duong ◽  
James Lim ◽  
Vidyullatha Vasireddy ◽  
Tyler Papp ◽  
Hung Nguyen ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Types ◽  
Egfp Expression ◽  
Cell Type ◽  
Egfp Gene ◽  
Rat Cortical Neurons

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ acrossin vitroand ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

scholarly journals Complex II subunit SDHD is critical for cell growth and metabolism, which can be partially restored with a synthetic ubiquinone analog

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Aloka B. Bandara ◽  
Joshua C. Drake ◽  
David A. Brown
Keyword(s):  
Mammalian Cells ◽  
Krebs Cycle ◽  
Atp Synthesis ◽  
Hek293 Cells ◽  
Knockout Mutant ◽  
Complex Ii ◽  
Growth Defects ◽  
Transport Chain ◽  
Mutant Cells

Abstract Background Succinate dehydrogenase (Complex II) plays a dual role in respiration by catalyzing the oxidation of succinate to fumarate in the mitochondrial Krebs cycle and transferring electrons from succinate to ubiquinone in the mitochondrial electron transport chain (ETC). Mutations in Complex II are associated with a number of pathologies. SDHD, one of the four subunits of Complex II, serves by anchoring the complex to the inner-membrane and transferring electrons from the complex to ubiquinone. Thus, modeling SDHD dysfunction could be a valuable tool for understanding its importance in metabolism and developing novel therapeutics, however no suitable models exist. Results Via CRISPR/Cas9, we mutated SDHD in HEK293 cells and investigated the in vitro role of SDHD in metabolism. Compared to the parent HEK293, the knockout mutant HEK293ΔSDHD produced significantly less number of cells in culture. The mutant cells predictably had suppressed Complex II-mediated mitochondrial respiration, but also Complex I-mediated respiration. SDHD mutation also adversely affected glycolytic capacity and ATP synthesis. Mutant cells were more apoptotic and susceptible to necrosis. Treatment with the mitochondrial therapeutic idebenone partially improved oxygen consumption and growth of mutant cells. Conclusions Overall, our results suggest that SDHD is vital for growth and metabolism of mammalian cells, and that respiratory and growth defects can be partially restored with treatment of a ubiquinone analog. This is the first report to use CRISPR/Cas9 approach to construct a knockout SDHD cell line and evaluate the efficacy of an established mitochondrial therapeutic candidate to improve bioenergetic capacity.

scholarly journals Position Effects Are Influenced by the Orientation of a Transgene with Respect to Flanking Chromatin

2001 ◽  
Vol 21 (1) ◽  
pp. 298-309 ◽  
Author(s):  
Yong-Qing Feng ◽  
Matthew C. Lorincz ◽  
Steve Fiering ◽  
John M. Greally ◽  
Eric E. Bouhassira
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Methylation Status ◽  
Regulatory Elements ◽  
Cell Populations ◽  
Methylation Analysis ◽  
Position Effects ◽  
Methylation State ◽  
Cassette Exchange

ABSTRACT We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

scholarly journals Evaluation of Transduction Properties of an Adenovirus Vector in Neonatal Mice

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Shunsuke Iizuka ◽  
Fuminori Sakurai ◽  
Kahori Shimizu ◽  
Kazuo Ohashi ◽  
Shin-ichiro Nakamura ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Adenovirus Vector ◽  
Firefly Luciferase ◽  
Luciferase Expression ◽  
Cmv Promoter ◽  
Neonatal Mice ◽  
Neonatal Heart ◽  
Neonatal Liver ◽  
Neonates And Infants

In gene therapy for congenital disorders, treatments during neonate and infant stages are promising. Replication-incompetent adenovirus (Ad) vectors have been used in gene therapy studies of genetic disorders; however, the transduction properties of Ad vectors in neonates and infants have not been fully examined. Accordingly, this study examined the properties of Ad vector-mediated transduction in neonatal mice. A first-generation Ad vector containing a cytomegalovirus (CMV) promoter-driven luciferase expression cassette was administered to neonatal mice on the second day of lifeviaretro-orbital sinus. The highest Ad vector genome copy numbers and transgene expression were found in the neonatal liver. The neonatal heart exhibited the second highest levels of transgene expression among the organs examined. There was an approximately 1500-fold difference in the transgene expression levels between the adult liver and heart, while the neonatal liver exhibited only an approximately 30-fold higher level of transgene expression than the neonatal heart. A liver-specific promoter for firefly luciferase expression conferred a more than 100-fold higher luciferase expression in the liver relative to the other organs. No apparent hepatotoxicity was observed in neonatal mice following Ad vector administration. These findings should provide valuable information for gene therapy using Ad vectors in neonates and infants.

scholarly journals Two Different Transgenes to Study Gene Silencing and Re-Expression During Zebrafish Caudal Fin and Retinal Regeneration

2006 ◽  
Vol 6 ◽  
pp. 65-81 ◽  
Author(s):  
Ryan Thummel ◽  
Christopher T. Burket ◽  
David R. Hyde
Keyword(s):  
Tissue Regeneration ◽  
Transgene Expression ◽  
Egfp Expression ◽  
Transgenic Zebrafish ◽  
Retinal Regeneration ◽  
Caudal Fin ◽  
Fin Regeneration ◽  
Differentiated Cells ◽  
Neuronal Progenitors ◽  
Wound Epithelium

We used the 500-bpXenopusef1-α promoter and the 2-kb zebrafish histone2A.F/Zpromoter to generate several independent transgenic zebrafish lines expressing EGFP. While both promoters drive ubiquitous EGFP expression in early zebrafish development, they are systematically silenced in several adult tissues, including the retina and caudal fin. However, EGFP expression is temporarily renewed in the adult during either caudal fin or retinal regeneration. In the Tg(H2A.F/Z:EGFP)ntline, EGFP is moderately expressed in both the wound epithelium and blastema of the regenerating caudal fin. In the Tg(ef1-α:EGFP)ntline, EGFP expression is reinitiated and restricted to the blastema of the regenerating caudal fin and colabels with BrdU, PCNA, andmsxc-positive cells. Thus, these two ubiquitous promoters drive EGFP transgene expression in different cell populations during caudal fin regeneration. We further analyzed the ability of theef1-α:EGFPtransgene to label nonterminally differentiated cells during adult tissue regeneration. First, we demonstrated that the transgene is highly methylated in adult zebrafish caudal fin tissue, but not during fin regeneration, implicating methylation as a potential means of transgene silencing in this line. Next, we determined that theef1-α:EGFPtransgene is also re-expressed during adult retinal regeneration. Specifically, theef1-α:EGFPtransgene colabels with PCNA in the Müglia, a specialized cell that is the source of neuronal progenitors during zebrafish retinal regeneration. Thus, we concluded that Tg(ef1-α:EGFP)nt line visually marks nonterminally differentiated cells in multiple adult regeneration environments and may prove to be a useful marker in tissue regeneration studies in zebrafish.

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