Comparative evaluation of the transgene expression efficiency provided by the model genetic constructs of different structure

Komissarov, Alexey A.; Karaseva, Maria A.; Safina, Dina; Roshchina, M. P.; Bednova, O. P.; Kazakov, A. A.; Демкин, В. В.; Demidiuk, I. V.

doi:10.18821/0208-0613-2016-34-3-115-120

Cited by 3 publications

(4 citation statements)

References 19 publications

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“…Two genetic constructs were derived from the pCI vector: pCI-3C for the constitutive expression of active hepatitis A virus 3C protease (3Cpro) and pCI-3Cmut expressing the mutant enzyme with no proteolytic activity due to the Cys172-Ala substitution in the active site (3Cmut) ( Figure 1 A) [ 13 , 14 ]. HEK293, HeLa, and A549 cells were transfected with these genetic constructs, and 15 h post transfection (p.t.…”

Section: Resultsmentioning

confidence: 99%

“…The proportion of cells expressing the target transgene post transfection is commonly used to evaluate the transfection agent efficiency; this parameter is often referred to as “transfection efficiency”. Our experiments with EGFP as a reporter indicated that the transfection efficiency of the used transfection agents does not usually exceed 40–50% for HEK293 cells and is much lower, ≈10–20%, for HeLa and A549 cells [ 13 ]. However, here, we have found out that the proportion of dead cells is much higher than the mean transfection efficiency after transient transfection (about 80% for HEK293 and over 95% for HeLa and A549).…”

Section: Discussionmentioning

confidence: 99%

“…The fragment was purified using a Cleanup Standard kit (Evrogen, Moscow, Russia), digested with EcoRI and KpnI enzymes (SibEnzyme, Novosibirsk, Russia) and cloned into pCI (Promega, Madison, WI, USA) digested with the same enzymes. Plasmid pCI-3Cmut was constructed in the same way except that pBI-EGFP-3Cmut [ 11 ] was the source of the 3Cmut gene encoding inactivated 3Cpro with the Cys172-Ala substitution; pCI-EGFP was constructed previously [ 13 ]. All plasmids were amplified in E. coli TG1 cells and purified using a Plasmid Miniprep kit (Evrogen, Moscow, Russia).…”

Section: Methodsmentioning

confidence: 99%

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Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

Komissarov

Karaseva

Roschina

et al. 2021

IJMS

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Regulated cell death (RCD) is a fundamental process common to nearly all living beings and essential for the development and tissue homeostasis in animals and humans. A wide range of molecules can induce RCD, including a number of viral proteolytic enzymes. To date, numerous data indicate that picornaviral 3C proteases can induce RCD. In most reported cases, these proteases induce classical caspase-dependent apoptosis. In contrast, the human hepatitis A virus 3C protease (3Cpro) has recently been shown to cause caspase-independent cell death accompanied by previously undescribed features. Here, we expressed 3Cpro in HEK293, HeLa, and A549 human cell lines to characterize 3Cpro-induced cell death morphologically and biochemically using flow cytometry and fluorescence microscopy. We found that dead cells demonstrated necrosis-like morphological changes including permeabilization of the plasma membrane, loss of mitochondrial potential, as well as mitochondria and nuclei swelling. Additionally, we showed that 3Cpro-induced cell death was efficiently blocked by ferroptosis inhibitors and was accompanied by intense lipid peroxidation. Taken together, these results indicate that 3Cpro induces ferroptosis upon its individual expression in human cells. This is the first demonstration that a proteolytic enzyme can induce ferroptosis, the recently discovered and actively studied type of RCD.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

Komissarov

Karaseva

Roschina

et al. 2021

IJMS

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show abstract

“…The fragment was purified using a Cleanup Standard kit (Evrogen, Russia), digested with Eco RI and Kpn I enzymes (SibEnzyme, Russia), and cloned into pCI (Promega, USA) digested with the same enzymes. Plasmid pCI-3Cmut was constructed in the same way except that pBI-EGFP-3Cmut [11] was the source of the 3Cmut gene encoding inactivated 3Cpro with the Cys172-Ala substitution; pCI-EGFP was constructed previously [13]. All plasmids were amplified in E. coli TG1 cells and purified using a Plasmid Miniprep kit (Evrogen, Russia).…”

Section: Methodsmentioning

confidence: 99%

Individual expression of hepatitis A virus 3C protease induces ferroptosis in human cellsin vitro

Komissarov

Karaseva

Roschina

et al. 2021

Preprint

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Functional efficiency of PCR vectors in vitro and at the organism level

et al. 2020

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The functional efficiency of the expression cassettes integrated into a plasmid and a PCRamplified fragment was comparatively analyzed after transient transfection in vitro or introduction into the developing embryo of Danio rerio. The cassettes contained the reporter genes, luciferase of Photinus pyralis (luc) or enhanced green fluorescent protein, under the control of the promoter of human cytomegalovirus immediate-early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times higher than that of the PCR-amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR-amplified fragment was quantitatively evaluated. The mutations generated after 25 amplification cycles with Taq DNA polymerase decreased luciferase activity in transfected cells by 65-85%. Thus, mutations are the key factor of decreased functional efficiency of the PCR-amplified fragment relative to the circular plasmid in this experimental model, while other factors apparently have a lesser impact. At the organism level, no significant difference in the expression efficiency of the plasmid and PCR-amplified fragment has been revealed. Comparison of the vector efficiencies in in vivo and in vitro systems demonstrates that the level of luciferase in the D. rerio cell lysate, normalized to the molar concentration of the vector, is by three orders of magnitude higher than that after the cell transfection in vitro, which indicates that the quantitative data obtained for in vitro systems should not be directly extrapolated to the organism level.

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Comparative evaluation of the transgene expression efficiency provided by the model genetic constructs of different structure

Cited by 3 publications

References 19 publications

Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

Individual Expression of Hepatitis A Virus 3C Protease Induces Ferroptosis in Human Cells In Vitro

Individual expression of hepatitis A virus 3C protease induces ferroptosis in human cellsin vitro

Functional efficiency of PCR vectors in vitro and at the organism level

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