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About 835 papers for “Nucleofection”

Nucleofection

The Dictionary of Genomics, Transcriptomics and Proteomics · 2015

Cited by 0

Amaxa nucleofection assays.

Virginie Coustou (252355), Fabien Guegan (252358), Nicolas Plazolles (252362) et al. · Figshare · 2015

<p>Once selected, transfectants were analyzed by PCR on genomic DNA, observation of EGFP fluorescence, or Renilla luciferase activity measurement.</p>

Cited by 0Open access

Days 0: Nucleofection

2016

Two days before Nucleofection split LCL line into 2xT25 flasks containing 5-10 mL per flask at a density of 0.35x106 cells/mL. If the line tends to grow slower, dilute to a cell concentration such that the cells will reach a density of 0.8-1x106 cells by the day of Nucleofection.

Cited by 0

Optimization of nucleofection conditions.

Wu Ou (491021), Pingjuan Li (491022), Jakob Reiser (491023) · Figshare · 2015

<p>A. Percentage of GFP-positive cells. One million DF19 cells were transfected with 2 µg of the pmaxGFP plasmid using the Nucleofector 2b device and different Nucleofection solutions and programs. Two days post transfection, the percentage of GFP positive cells was determined using FACS. B. FACS profile of cells nucleofected using Solution I and program B016. The gray line represents transfected cells and the black graph untransfected cells. C. Cell viability. Cell viability was determined using Promega's CellTiter-Glo kit. The viability of control cells that were not nucleofected but otherwise treated in the same way was taken as 100%.</p>

Cited by 0Open access

Nucleofection of human iPSCs.

Koollawat Chupradit (11536487), Nontaphat Thongsin (12269593), Chatchai Tayapiwatana (88185) et al. · Figshare · 2022

<p>(A) Schematic diagram of the gene-editing strategy targeting the <i>AAVS1</i> locus using the CRISPR/Cas9 RNP complex and the recombinant AAV6 vectors in human iPSCs. HR = Homologous Recombination. (B) Microscopic fluorescence analysis of human iPSCs shows mCherry expression on days 1, 4 and day 6 post-nucleofection from three different conditions. Scale bar = 100 μm (Day 1) and 200 μm (Days 4 and 6). (C) The percentage of mCherry<sup>+</sup> cells on day 6 post-nucleofection as analyzed by flow cytometry.</p>

Cited by 0Open access

Nucleofection of hPSCs v1

Hanqin Li · 2022

This protocol describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection. General notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

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CD34+ Cell RNP Nucleofection v1

Daniel A. Kuppers · 2022

Human CD34+hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate “humanized mice”. To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34+HSPCs before transplantation. We have optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes and are now able to edit a candidate gene in CD34+HSPCs with almost 100% efficiency, and without affecting their potential for engraftment and multilineage differentiation in mice.

Cited by 0Open access

CD34+ Cell RNP Nucleofection v2

Daniel A. Kuppers · 2023

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) are a standard source of cells for clinical HSC transplantations as well as experimental xenotransplantation to generate “humanized mice”. To further extend the range of applications of these humanized mice, we developed a protocol to efficiently edit the genomes of human CD34+ HSPCs before transplantation. In the past, manipulating HSPCs has been complicated by the fact that they are inherently difficult to transduce with lentivectors, and rapidly lose their stemness and engraftment potential during in vitro culture. However, with optimized nucleofection of sgRNA:Cas9 ribonucleoprotein complexes, we are now able to edit a candidate gene in CD34+ HSPCs with almost 100% efficiency, and still be able to generate a humanized mouse with high engraftment and multilineage differentiation.

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Nucleofection of whole murine retinas

González García, Francisco · LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) · 2013

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3.2 Nucleofection of iPSCs v1

Evelyn J. Sauter, Lisa K. Kutsche, Simon D. Klapper et al. · 2020

This is part 3.2 of the "Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders" collection of protocols. Collection Abstract: Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.

Cited by 0Open access

Nucleofection of Human Embryonic Stem Cells

Henrike Siemen, Michael Nix, Elmar Endl et al. · Stem Cells and Development · 2005

Human embryonic stem (hES) cells provide an important tool for the study of human development, disease, and tissue regeneration. Technologies for efficient genetic modification are required to exploit hES cells fully for these applications. Here we present a customized protocol for the transfection of hES cells with the Nucleofector technology and compare its efficiency with conventional electroporation and lipofection. Cell survival and transfection efficiency were quantified using an enhanced green fluorescent protein (EGFP) reporter construct. Our optimized nucleofection parameters yielded survival rates >70%. Under these conditions, 66% of the surviving cells showed transgene expression 24 h after nucleofection. Transfected cells maintained expression of the pluripotency- associated markers Tra-1-60, Tra-1-81, and Oct4 and could be expanded to stably transgene-expressing clones. The low quantities of hES cells and DNA required for nucleofection could make this method an attractive tool for miniaturized high throughput screening (HTS) applications.

Cited by 89

Nucleofection ® of mouse ES cells

Sabine Boljahn, Anja Rode, Maria Joao da Silva et al. · 2004

Gene targeting in embryonic stem (ES) cells relies on the homologous recombination (HR) of a native chromosomal gene and introduced exogenous DNA [1-3]. Conditional mutagenesis of mice is a highly sophisticated variation of gene targeting, generating a ‘switch’ that allows genes to be activated or silenced at will. Via HR, a silent mutation in a gene of interest is created by flanking essential parts of a gene with two recombination recognition sites (RRS). Time or tissue-specific expression of a specific recombinase allows deletion of the intervening sequence between two RRS, and creation of the mutant genotype. The two most widely applied are the Cre – loxP and the Flp – frt systems [4]. A combination of both systems is often employed to remove the antibiotic resistance gene cassette which is required for selection of ES cell clones, but may interfere with gene expression. The respective recombinase is expressed transiently in targeted ES cells in a second transfection round, and deleted (antibiotic sensitive) clones selected by single cell plating and subsequent molecular analysis. Gene targeting requires transfection of the foreign DNA into the ES cells, traditionally performed by electroporation. Nucleofection is a new method mediating high transfection efficiencies in primary cells due to direct delivery of the DNA into the nucleus. We were interested in studying the effects of nucleofection on homologous recombination and transient transfection in ES cells. Therefore we directly compared standard electroporation and nucleofection in our conditional mutagenesis projects. The pluripotent quality of nucleofected ES cells was assayed by the generation of chimeras via diploid blastocyst injection or ES-derived mice via tetraploid complementation. The latter are mice derived solely from embryonic stem cells [5, 6, 7].

Cited by 0

Nucleofection of Human Embryonic Stem Cells

Henrike Siemen, Lars Nolden, Stefanie Terstegge et al. · Methods in molecular biology · 2008

Human embryonic stem cells (HESCs) are widely used as a model system for human cell type specification. Genetic modification forms a valuable tool for HESC technology, as it provides the basis for lineage selection, i.e., the purification of a specific cell type after differentiation. Electroporation is an efficient way to transfect HESCs. Nucleofection is an electroporation-based transfection technique which utilizes cell-type-specific buffer solutions and specific electric settings. Customization of these two parameters has been proven to result in highly efficient gene transfer even in hard-to-transfect cells. We can show that nucleofection surpasses conventional electroporation in efficiency and decreases the experimental effort for transfection of HESCs.

Cited by 4

Nucleofection of Human Embryonic Stem Cells

Helen Fong, Kristi Elliott, Leslie F. Lock et al. · Methods in molecular biology · 2011

The ability to realize the full potential of human pluripotent stem cells (hPSCs) as tools for -understanding human development and advancing the field of regenerative medicine is dependent on efficient methods to genetically manipulate these cells. There are several methods for introducing foreign DNA into cells such as electroporation, lipid-based transfection technology, and viral transduction. We describe here a method to transfect human embryonic stem cells (hESCs) using nucleofection technology. This unique method uses the Nucleofector II Device that combines the use of a cell type-specific Nucleofector Solution and preprogrammed electrical parameters to efficiently deliver DNA into the cell nucleus. The use of this technology allows high-efficiency transfer of nucleic acids into hESCs enabling both transient and stable manipulation of gene expression in these cells.

Cited by 7

Quantitative RT-PCR post nucleofection / incubation.

Yann Duchartre (4579672), Stefanie Bachl (4579681), Hye Na Kim (4579669) et al. · Figshare · 2017

<p><b>A.</b> Expression of CD49d in Kasumi-2 cells nucleofected with ATL1102 after 24h, 48h and 72h. <b>B.</b> Expression of CD49d in Kasumi-2 cells incubated with ATL1102 after 48h (n = 1). For technical reasons, RT-PCR for incubated cells was only performed once but observations agreed with the concurrently generated flow cytometry data as well as with the data generated by electroporating with ATL1102.</p>

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