An Efficient Method for Isolation of Plasmid DNA for Transfection of Mammalian Cell Cultures

Kachkin, Daniel V.; Khorolskaya, Julia I.; Ivanova, Julia; Rubel, Aleksandr A.

doi:10.3390/mps3040069

Cited by 3 publications

(4 citation statements)

References 12 publications

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“…To make L-MSCs-EGFP, a lentiviral vector encoding the fluorescent protein EGFP sequence controlled by the constitutive CMV promoter was preliminarily obtained [ 23 ]. This lentiviral vector was used to transduce L-MSCs isolated from the rabbit limbus.…”

Section: Resultsmentioning

confidence: 99%

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Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology

et al. 2021

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The development of cell-based approaches to the treatment of various cornea pathologies, including limbal stem cell deficiency (LSCD), is an area of current interest in regenerative biomedicine. In this context, the shortage of donor material is urgent, and limbal mesenchymal stem cells (L-MSCs) may become a promising cell source for the development of these novel approaches, being established mainly within the rabbit model. In this study, we obtained and characterized rabbit L-MSCs and modified them with lentiviral transduction to express the green fluorescent protein EGFP (L-MSCs-EGFP). L-MSCs and L-MSCs-EGFP express not only stem cell markers specific for mesenchymal stem cells but also ABCG2, ABCB5, ALDH3A1, PAX6, and p63a specific for limbal epithelial stem cells (LESCs), as well as various cytokeratins (3/12, 15, 19). L-MSCs-EGFP have been proven to differentiate into adipogenic, osteogenic, and chondrogenic directions, as well as to transdifferentiate into epithelial cells. The possibility of using L-MSCs-EGFP to study the biocompatibility of various scaffolds developed to treat corneal pathologies was demonstrated. L-MSCs-EGFP may become a useful tool for studying regenerative processes occurring during the treatment of various corneal pathologies, including LSCD, with the use of cell-based technologies.

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Section: Resultsmentioning

confidence: 99%

“…To make a cell culture of rabbit L-MSCs labeled with a green fluorescent protein EGFP, a lentiviral vector LV-CMV-EGFP Hygro (656-4) was generated, as previously described [ 23 ]. The basic plasmid pLenti-CMV-EGFP Hygro (656-4) [ 24 ] was used for the production of the second-generation lentivirus assembly system.…”

Section: Methodsmentioning

confidence: 99%

Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology

et al. 2021

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“…Plasmid DNA construction was performed according to the standard protocols described by Sambrook et al [ 59 ]. Plasmid extraction and purification from E. coli were conducted according to the procedures described by Kachkin et al [ 60 ]. Yeast DNA transformations were performed by a protocol involving lithium acetate treatment and heat shock [ 61 ].…”

Section: Methodsmentioning

confidence: 99%

The Aβ42 Peptide and IAPP Physically Interact in a Yeast-Based Assay

Kachkin,

Lashkul,

Gorsheneva

et al. 2023

IJMS

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Numerous studies have demonstrated that people with type 2 diabetes mellitus (associated with IAPP peptide aggregation) show an increased incidence of Alzheimer’s disease (associated with Aβ aggregation), but the mechanism responsible for this correlation is presently unknown. Here, we applied a yeast-based model to study the interactions of IAPP with PrP (associated with TSEs) and with the Aβ42 peptide. We demonstrated that fluorescently tagged IAPP forms detergent-resistant aggregates in yeast cells. Using the FRET approach, we showed that IAPP and Aβ aggregates co-localize and physically interact in yeast cells. We also showed that this interaction is specific and that there is no interaction between IAPP and PrP in the yeast system. Our data confirmed a direct physical interaction between IAPP and Aβ42 aggregates in a living cell. Based on these findings, we hypothesize that this interaction may play a crucial role in seeding Aβ42 aggregation in T2DM patients, thereby promoting the development of AD.

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“…The cells were incubated for 18 h at 37 °C in a CO 2 incubator with 5% CO 2 in humidified conditions. After 18 h, the medium with the transfection mixture was removed and fresh standard medium was added [ 74 ].…”

Section: Methodsmentioning

confidence: 99%

Human RAD51 Protein Forms Amyloid-like Aggregates In Vitro

Kachkin

Volkov

Sopova

et al. 2022

IJMS

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RAD51 is a central protein of homologous recombination and DNA repair processes that maintains genome stability and ensures the accurate repair of double-stranded breaks (DSBs). In this work, we assessed amyloid properties of RAD51 in vitro and in the bacterial curli-dependent amyloid generator (C-DAG) system. Resistance to ionic detergents, staining with amyloid-specific dyes, polarized microscopy, transmission electron microscopy (TEM), X-ray diffraction and other methods were used to evaluate the properties and structure of RAD51 aggregates. The purified human RAD51 protein formed detergent-resistant aggregates in vitro that had an unbranched cross-β fibrillar structure, which is typical for amyloids, and were stained with amyloid-specific dyes. Congo-red-stained RAD51 aggregates demonstrated birefringence under polarized light. RAD51 fibrils produced sharp circular X-ray reflections at 4.7 Å and 10 Å, demonstrating that they had a cross-β structure. Cytoplasmic aggregates of RAD51 were observed in cell cultures overexpressing RAD51. We demonstrated that a key protein that maintains genome stability, RAD51, has amyloid properties in vitro and in the C-DAG system and discussed the possible biological relevance of this observation.

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An Efficient Method for Isolation of Plasmid DNA for Transfection of Mammalian Cell Cultures

Cited by 3 publications

References 12 publications

Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology

Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology

The Aβ42 Peptide and IAPP Physically Interact in a Yeast-Based Assay

Human RAD51 Protein Forms Amyloid-like Aggregates In Vitro

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