nucleofection
25110540
Nucleofection with Plasmid DNA for CRISPR/Cas9 Mediated Inactivation of Programmed Cell Death Protein 1 in CD133 Specific CAR T Cells.
CRISPR/Cas9 mediated programmed cell death protein 1 (PD 1) disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in an immunosuppressive tumor microenvironment. In most of the reported cases, Cas9 was delivered into T cells by way of electroporation with RNA or protein. However, transient expression of Cas9 by transfection with a plasmid encoding its gene is apparently simpler, as it avoids the steps of in vitro transcription of DNA or protein production. This study tried nucleofection into human primary T cells of plasmids encoding both CRISPR/Cas9 for disrupting the PD 1 gene and the piggyBac transposon system for expressing CD133 specific CAR in one reaction. Based on drug selection, CD133 specific CAR T cells were obtained in which, on average, 91.5% of the PD 1 gene sites were disrupted, but almost no Cas9 gene expression was found in the final engineered CAR T cells. The PD 1 deficient CD133 specific CAR T cells showed similar levels of cytokine secretion and improved proliferation and cytotoxicity in vitro, and enhanced inhibition of tumor growth in an orthotopic mouse model of glioma, compared to conventional CD133 CAR T cells. The described method could be useful for the production of PD 1 deficient CAR T cells for cancer immunotherapy.
Human gene therapy
Bian Hu Yan Zou Linlin Zhang Jiaxing Tang Gabriele Niedermann Elke Firat Xingxu Huang Xuekai Zhu
211726452
Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites.
Transfection is a technical process through which genetic material, such as DNA and double stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.
Journal of visualized experiments JoVE
Chunhui Duan Xinming Tang Dandan Hu Sixin Zhang Jie Liu Feifei Bi Zhenkai Hao Jingxia Suo Yingchao Yu Min Wang Pei Sun Liying Du Xun Suo Xianyong Liu
221285843
Efficient Multi Allelic Genome Editing of Primary Cell Cultures via CRISPR Cas9 Ribonucleoprotein Nucleofection.
CRISPR Cas9 based technologies have revolutionized experimental manipulation of mammalian genomes. However, limitations regarding the delivery and efficacy of these technologies restrict their application in primary cells. This article describes a protocol for penetrant, reproducible, and fast CRISPR Cas9 genome editing in cell cultures derived from primary cells. The protocol employs transient nucleofection of ribonucleoprotein complexes composed of chemically synthesized 2' O methyl 3'phosphorothioate modified single guide RNAs (sgRNAs) and purified Cas9 protein. It can be used both for targeted insertion deletion mutation (indel) formation at up to >90% efficiency (via use of a single sgRNA) and for targeted deletion of genomic regions (via combined use of multiple sgRNAs) This article provides examples of the nucleofection buffer and programs that are optimal for patient derived glioblastoma (GBM) stem like cells (GSCs) and human neural stem/progenitor cells (NSCs) but the protocol can be readily applied to other primary cell cultures by modifying the nucleofection conditions. In summary, this is a relatively simple method that can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells such as many cancer stem like cells. (c) 2020 Wiley Periodicals LLC Basic Protocol: Cas9:sgRNA ribonucleoprotein nucleofection for insertion deletion (indel) mutation and genomic deletion generation in primary cell cultures.
Current protocols in stem cell biology
Pia Hoellerbauer Megan Kufeld Patrick J Paddison
222236266
Chemically modified mRNA nucleofection of primary human T cells.
Here we show that an approach of in vitro transcribed mRNA nucleofection expands the range of transfection of primary human T cells. It represents a reproducible and time efficient technology, and is thus an ideal tool in basic research involving highly controlled in vitro experiments with a gene of interest aiming at identifying its biological human T cell function.
Journal of immunological methods
Nikolaus Thuille Tajana Sajinovic Kerstin Siegmund Gottfried Baier
218780582
Effective Lonza 4D Nucleofection with Inexpensive Homemade Buffers v1
This protocol uses the Lonza 4D nucleofection system. Here I present protocols that reduce the price dramatically of using Lonza 4D equipment. 1. The 16 well strip kits are expensive. These can be washed and re used. This allows a single strip for hundreds of nucleofection experiments. 2. The nucleofection buffers are limited (P1, P2, etc) However we have used home made buffers that effectivly target many primary cell types. We have used this Cas9 RNPs (protein/RNA) and plasmid delivery. We have used this in T and B cells, cell lines, and more. The homemade buffer used here was originally used and published in: https:/cancerres.aacrjournals.org/content/71/23/7291. It was called Amaxa V. The actual recipe used in that paper was published in the thesis here: https:/opus4.kobv.de/opus4 fau/frontdoor/index/index/docId/2189 Here we show a simple nucleofection of plasmid into K562. THIS PROTOCOL ACCOMPANIES THE FOLLOWING PUBLICATION test DOI dx.doi.org/10.17504/protocols.io.64mhgu6 PROTOCOL CITATION Eric Danner 2020. Effective Lonza 4D Nucleofection with Inexpensive Homemade Buffers protocols.io https:/dx.doi.org/10.17504/protocols.io.64mhgu6 MANUSCRIPT CITATION please remember to cite the following publication along with this protocol test
protocols.io
Eric W Danner
216593616
Nucleofection of phiC31 Integrase Protein Mediates Sequence Specific Genomic Integration in Human Cells.
The phage derived phiC31 integrase is a useful tool for mediating sequence specific genomic integration in mammalian cells, recombining donor plasmids bearing the attB recognition site with introduced genomic attP sites or endogeneous pseudo attP sites having partial identity to attP. In most prior studies, phiC31 integrase has been introduced as plasmid DNA or mRNA. The current report examines whether phiC31 integrase functions efficiently in mammalian cells when co nucleofected as a purified protein, along with attB containing donor plasmids or PCR fragments. We describe preparation of phiC31 integrase protein and evidence that it can mediate genomic integration in human 293 cells, including PCR evidence for integration at an endogenous pseudo attP site. This work demonstrates for the first time the ability of 605 and 613 amino acid versions of phiC31 integrase protein to mediate efficient, site specific integration into the genome of human cells when co nucleofected with full sized attB containing donor plasmids or linear 2.5 kb PCR fragments. This protein mediated approach may be especially useful for integration of exogenous sequences into valuable therapeutic target cells, such as hematopoietic stem cells or T cells, that are sensitive to introduced DNA.
Journal of molecular biology
Tuhin Kumar Guha Michele P Calos
58591607
Stable transfection of Eimeria necatrix through nucleofection of second generation merozoites.
Eimeria spp. the causative agents of coccidiosis, are the most common protozoan pathogens of chickens. Infection with these parasites can result in poor development or death of animals leading to a devastating economic impact on poultry production. The establishment of transfection protocols for genetic manipulation of Eimeria species and stable expression of genes would help advance the biology of these parasites as well as establish these organisms as novel vaccine delivery vehicles. Here, we report the selection of the first stable transgenic E. necatrix population, EnHA1, consitutively expressing the EYFP reporter following transfection of the 2nd generation merozoites with a linear DNA fragment harboring the EYFP reporter gene, the HA1 gene from the avian influenza virus H9N2 and the TgDHFR TS selectable marker, which confers resistance to pyrimethamine. Transfected merozoites were inoculated into chickens via the cloacal route, and feces from 18 h to 72 h post inoculation were collected and subjected to subsequent serial passages, FACS sorting and pyrimethamine selection. A gradual increase in the number of EYFP expressing sporulated oocysts was noticed with more than 90% EYFP oocysts obtained after five passages. Immunofluorescence assay confirmed successful expression of the HA1 antigen in the EnHA1 population. The ability to genetically manipulate E. necatrix merozoites and express heterologous genes in this parasite will pave the way for possible use of this organism as a vaccine delivery vehicle.
Molecular and biochemical parasitology
Chunhui Duan Dandan Hu Xinming Tang Jingxia Suo Si Wang Sixin Zhang Geru Tao Chao Li Chaoyue Wang Xiaolong Gu Xiaoli Tang Guangping Huang Bi Xiang Shaoqiang Wu Choukri Ben Mamoun Xun Suo Xianyong Liu
116241516
Nucleofection with Plasmid DNA for CRISPR/Cas9 Mediated Inactivation of Programmed Cell Death Protein 1 in CD133 Specific CAR T Cells
CRISPR/Cas9 mediated programmed cell death protein 1 (PD 1) disruption in chimeric antigen receptor (CAR) T cells could be an appealing choice to improve the therapeutic efficacy of CAR T cells in
90763534
Polyplex System Versus Nucleofection for Human Skin Cell Transfection and Effect of Internal Ribosome Entry Site Sequence
Nonviral transfection has important implications on gene therapy because of its safety. In particular, polyfection and nucleofection are two widely used systems for nonviral gene delivery. Their potential depends on the transfection efficiency achieved, which is influenced in turn by the type of cells transfected and by the plasmid that carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblasts and keratinocytes was evaluated in this study. Transfections were performed with plasmids containing a gene of interest (human cathelicidin antimicrobial peptide) and two reporter genes (red or green fluorescent protein) that included or not an internal ribosome entry site (IRES) The efficiency was measured by flow cytometry in terms of percentage of cells expressing the reporter gene; viability of transfected cells was also evaluated. It was found that nucleofection was more efficient than polyplexes for transfecting fibroblasts, while no significant differences were found between both systems of transfection when applied to keratinocytes. Regarding the viability of fibroblasts after transfection, values were high in both systems. In contrast, keratinocytes were more sensitive to nucleofection. It was also noted that both types of cells decreased reporter gene expression when IRES sequence was located upstream of the reporter gene, suggesting a negative effect on the expression of this gene. These results confirm that the transfection efficiency depends on the type of cells and the system used.
Natalia Yiset Becerra Colorado Claudia Marcela Arenas Gomez Maria Isabel Patino Vargas Jean Paul Delgado Charris Carlos Enrique Muskus Lopez Luz Marina Restrepo Munera
4861310
Polyplex System Versus Nucleofection for Human Skin Cell Transfection and Effect of Internal Ribosome Entry Site Sequence.
Nonviral transfection has important implications on gene therapy because of its safety. In particular, polyfection and nucleofection are two widely used systems for nonviral gene delivery. Their potential depends on the transfection efficiency achieved, which is influenced in turn by the type of cells transfected and by the plasmid that carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblasts and keratinocytes was evaluated in this study. Transfections were performed with plasmids containing a gene of interest (human cathelicidin antimicrobial peptide) and two reporter genes (red or green fluorescent protein) that included or not an internal ribosome entry site (IRES) The efficiency was measured by flow cytometry in terms of percentage of cells expressing the reporter gene; viability of transfected cells was also evaluated. It was found that nucleofection was more efficient than polyplexes for transfecting fibroblasts, while no significant differences were found between both systems of transfection when applied to keratinocytes. Regarding the viability of fibroblasts after transfection, values were high in both systems. In contrast, keratinocytes were more sensitive to nucleofection. It was also noted that both types of cells decreased reporter gene expression when IRES sequence was located upstream of the reporter gene, suggesting a negative effect on the expression of this gene. These results confirm that the transfection efficiency depends on the type of cells and the system used.
Tissue engineering. Part C, Methods
Natalia Yiset Becerra Colorado Claudia Marcela Arenas Gomez Maria Isabel Patino Vargas Jean Paul Delgado Charris Carlos Enrique Muskus Lopez Luz Marina Restrepo Munera