is a Parker Institute for Cancer Immunotherapy member. Materials Protocol Data Materials Eyquem, J. et al. Electroporate the cells, recover the strip and turn off the instrument.
The effects of electroporation buffer composition on cell - Nature J. Immunol. PubMed Central Further development of electroporation by legacy Amaxa has led to the development of a more efficient transfection technology called Nucleofection. One method to generate long ssDNA templates involves a two-step selective exonuclease digestion that specifically degrades one strand of a PCR product that has been labelled by 5 phosphorylation, which can be easily added to a PCR primer before amplification. High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. This includes introducing new genes to study or leverage their function, as well as using other constructs to indirectly influence endogenous gene expression or other cellular processes. Primary human T cells with two modifications were enriched by gating on the cells that had at least one modification, and this effect was consistent across multiple combinations of genomic loci. Methodological changes that maximized efficiency were not always optimal for the total number of positive cells, and vice-versa. Bioz Stars score: 86/100, based on 1 PubMed citations. and A.M. designed the study and wrote the manuscript. Furthermore, with DNA-sensitive cells, cell viability is often much better when using RNA. Below, we outline the difference between stable and transient transfection and discuss the different transfection substrates you might use. Viability was measured 2days after electroporation and GFP expression was measured at day4. a, Histograms of IL-2R surface expression in CD3+ T cells in all children from a family carrying two loss-of-function IL2RA mutations, including three compound heterozygotes that express minimal amounts of IL-2R on the surface of the T cells (no electroporation, grey). In these experiments, expansion was less robust for cells from compound het 3. f, After 12 days in culture, corrected Treg cells from compound heterozygote 2, and a female healthy control, were sorted based on IL-2R and CD62L expression.
Nucleofector Technology - Stretching Transfection Dimensions - Lonza Quadros, R. M. et al. & Restifo, N. P. Adoptive cell transfer as personalized immunotherapy for human cancer. Ther. 4f). Have you considered all the practical issues (e.g. Well, the ideal approach should offer high transfection efficiency, low toxicity and yield reproducible results. As a maximum of two targeted insertions are possible (at the two alleles of the locus; assuming a diploid genome), no cells positive for all three loci should be observed (triple positives). After 12days in culture, during which time the cells expanded more than 100-fold, greater than 20% (compound het 1) and 40% (compound het 2) of targeted cells expressed IL-2R on their surface, demonstrating functional correction and expansion of a therapeutically relevant cell type. Primary human T cells edited to express an NY-ESO-1 antigen-specific TCR were generated (either by lentiviral transduction or non-viral TCR replacement), expanded for 10days after transduction or electroporation, and frozen. what is the size of your construct)? ORDER YOUR CUSTOM HDR TEMPLATE Guide RNAs Cas9 nucleases Enhancers & controls CRISPR-Cas9 gRNA Guide RNAs (gRNAs) contain the target-specific sequence for guiding Cas9 protein to a genomic location. RNP + HDRT; 2. Mix gently each sample and transfer 25 L in a free well of the 16-well electroporation strip (Lonza).
Frontiers | An Efficient Electroporation Protocol for the Genetic e, Note, with the high concentration of dsDNA used in these experiments, viability was higher if the RNP and cells were mixed first and the DNA template was added immediately before electroporation (1. Type in Product name, Keyword or Catalog number to see suggestions. RAB11AGFP template was used with on-target gRNA was used in ad. Electroporation is a physical transfection method that permeabilizes the cell membrane by applying an electrical pulse and moves molecules via the electrical field into the cell. A PCR product with a short T7 promoter appended serves as an IVT template to produce a ssRNA product. Graphs display mean and s.d. 5 x 106 NKL cells were transfected with 2.5 g of pmaxGFP Vector. dsDNA HDR temple (5 g) was used in ce. performed amplicon sequencing. When the mixture is incubated at room temperature, DNA-calcium phosphate coprecipitates form. 180, 61166131 (2008). Alexander Marson. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. CAS For ssODN electroporations, 100 pmol in 1 l water was electroporated. performed and analysed CUT&RUN experiments. As nucleic acids such as DNA and RNA are also negatively charged, they repel each other, inhibiting their uptake by the cell. ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells, Scalable continuous-flow electroporation platform enabling T cell transfection for cellular therapy manufacturing, Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells. 62/578,153,T.L.R., C.P.S., E.S., A.R. For each electroporation, 400 ng of the plasmid vector pMaxGFP (Lonza) in a volume of 2 l was placed in one well of a 96-well . 168, 724740 (2017). Different types of cells can be transfected (such as primary cells, cell lines, and stem cells), making this a powerful tool for use across the life sciences. To help you select the optimal transfection tool, consider these key questions: To further help you select the best way to transfect your cells, weve created a handy table comparing all the available transfection methods, highlighting their relative advantages and disadvantages (see below). Nat. This is a preview of subscription content, access via your institution. More details on each transfection substrate are provided below. Important note: The user bears the sole responsibility for determining the existence of any third party rights, as well as obtaining any necessary licenses. Skene, P. J. DNA, RNA, or protein) using one of various transfection methods, like lipofection, electroporation, calcium phosphate transfection, liposome transfection, and viral transduction, as well as physical methods such as microinjection and biolistic particle delivery. After annealing of an RT primer and reverse transcription, an RNADNA hybrid can form, which then can be transformed into a long ssDNA template by incubation in sodium hydroxide, which selectively degrades the RNA strand. Get the most important science stories of the day, free in your inbox. We offer 3 gRNA formats: crRNA:tracrRNA duplex, crRNA XT:tracrRNA duplex, and single guide RNA (sgRNA). f, Although efficient GFP expression depended on pairing the HDR template with the correct gRNA targeting that site, rare GFP+ cells were observed when dsDNA HDR templates were delivered either alone (~0.1%) or with an off-target Cas9 RNP (~1%). ), K23 DK094866 (S.W.G. PubMed Central In wells in which the RNP and the DNA HDR template were mixed together before adding the cells (1. Experiment in h was performed in two independent healthy donors. 506, 97114 (2009). This helps to ensure optimum transfection efficiency and cell viability. ad, Schematic description of HDR template for endogenous TCR replacement by in-frame integration of a new TCR- chain and a new variable region of a TCR- chain at the TCR- locus, and subsequent transcription and translation of the new TCR. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. and M.H. b, Targeting was similar either using bulk CD3+ T cells fresh after isolation or after cryopreservation (stored in liquid nitrogen and thawed before initial activation). The approach involves combining cationic lipids with other molecules to create unilamellar liposome vesicles that carry a positive charge. H.L., J.W. Stimulated Tresp cell population (solid curves), non-stimulated Tresp cells (dashed curve). ADS Nat. contributed to functional assays of edited T cells. Count cells using a hemocytometer or other automated method. and K.S. Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells.
Amaxa General Nucleofection Protocol - Lonza Article Stable transfection is often required for large-scale protein production, research into long-term gene regulation, the generation of stable cell lines, and for gene therapy. Schumann, K. et al. Natl Acad. A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Required Material The Lonza 4D-Nucleofector platform has multiple electroporation volume and vessel options. 191 talking about this. https://doi.org/10.1038/s41586-018-0326-5, DOI: https://doi.org/10.1038/s41586-018-0326-5. Nature 518, 337343 (2015). GFP mean fluorescent intensity (MFI) was calculated for the GFP+ cells in each condition/donor, and normalized as a percentage of the maximum GFP MFI observed. ISSN 1476-4687 (online) b, Amplicon sequencing was performed in select targeted patient cells. If youd like to learn more about Lipofection and other chemical-based transfection methods, download our Transfection Methods Technical Reference Guide here. Nature 543, 113117 (2017). Rev. performed or supervised in vitro assays of T cells with endogenous TCR knock-ins. Immunol. PBMCs were cultured for 2days identically to primary T cells (Methods). Mol. Across six unique HDR templates and gRNAs, on-target HDR-mediated integration was the by far most efficient. Plate cells to achieve 70-80% confluence the next day. A.M.F. T cells from two donors were each electroporated twice with an 8 h rest in between electroporations. XS Pichia and E.coli Expression Systems. 6i). 27 Iulie 2018. Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. Using this methodology, according to Nucleofactor . Open Access b, The frequency of one of the observed incorrect integrations at the target locus was reduced using a long ssDNA HDR template in two human blood donors (Supplementary Note2). Genetic and epigenetic fine mapping of causal autoimmune disease variants. On the day of electroporation, the gRNA (complexed with Cas9 to form an RNP), the HDR template, and collected stimulated T cells are mixed and electroporated, a process taking approximately 1.5h. After electroporation, engineered T cells can be readily expanded for an additional 12weeks. This research was supported by NIH grants DP3DK111914-01 (A.M.), P50GM082250 (A.M.), R35 CA197633 (A.R. n=2 (a, b, h, i) or n=8 (e, f) independent healthy donors. e, f, In these experiments, 17days after T cell transfer (d), non-virally TCR-replaced cells appeared to show greater NY-ESO-1 TCR expression and lower expression of exhaustion markers. T.L.R., R.Y., E.S., J.L., J.H., V.T., D.M.N. h, Arrayed testing of electroporation pulse conditions showed that, in general, conditions yielding higher HDR efficiency decreased viability. From a practical perspective, it should also be as quick and straightforward as possible to carry out. Google Scholar. Transfection is generally defined as the process of introducing DNA, RNA or proteins into cells to influence their genotype or phenotype. d, Electroporation of a ssDNA HDR template reduced off-target integrations to the limit of detection (that is, comparable to levels seen with no template electroporated) both with no nuclease added and at induced off-target dsDNA breaks (off-target gRNA + Cas9). Before choosing a transfection method, it is important to consider what substrate to transfect and whether you wish to achieve transient or stable transfection. Thank you for visiting nature.com. So I am trying to electroporate human dermal fibroblasts using the Lonza 4D nucleofector however the electroporation program that lonza recommends gives less than 20% GFP positive cells following .
Electroporation Cuvette | Lonza | Bioz Incubate cells at 37 C in 5% CO 2overnight. Cell densities greater than 80% may reduce electroporation efficiency. Mali, P., Esvelt, K. M. & Church, G. M. Cas9 as a versatile tool for engineering biology. This can include using DNA, proteins, mRNA or non-coding RNAs, all of which will degrade over time or will be diluted out through cell division. Cain-Hom, C. et al. Meanwhile, recombinant protein expression also enables a wide range of functional, genetic mapping and biochemical characterization studies, in addition to bioproduction applications. J. Immunol. The advantage of using proteins is that they usually exert their influence on the cell immediately and the quantity can be controlled much better. Preprint at https://doi.org/10.1101/178905 (2017). T.L.R. This toolbox of expression system components enables you to screen and make impactful choices.
Frontiers | Optimization of adult sensory neuron electroporation to RNP + Cells; 2. CAS c, pSTAT5 in response to high dose IL-2 stimulation (200 Uml1) in targeted CD3+ T cells after 7 days of expansion post-electroporation. d, Deficiency in IL-2R surface expression in compound heterozygote 3 led to aberrant downstream signalling as measured by pSTAT5 expression after stimulation with IL-2, but not IL-7 or IL-15. Although two closely interspersed electroporations caused a high degree of cell death, delivery of the RNP and linear dsDNA template could be delivered separately. 2. News Press Release Team Advisors Partners Contact Bioz Stars Bioz vStars 86 Buy from Supplier Structured Review Lonza p3 primary cell full electroporation buffer P3 Primary Cell Full Electroporation Buffer, supplied by Lonza, used in various techniques. 2 g of each template, along with 30 pmol of each associated RNP, were electroporated for dual multiplexing experiments. 4h. Small numbers of reads in the no electroporation and RNP only conditions were called as HDR, potentially owing to small amounts of cross-well contamination. One HDR template, a C-terminal GFP fusion tag into the nuclear factor FBL, had consistently higher off-target expression across gRNAs, potentially due to a gene-trap effect as the 3 homology arm for FBL contains a splice-site acceptor followed by the final exon of FBL leading into the GFP fusion. R.Y. Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. Nature (Nature) PubMed Bars represent observed GFP+ percentages from T cells from one representative donor electroporated with the indicated components. Starting with the bulk edited T cell population (all of which had been electroporated with the on-target gRNA and HDR template), we separately sorted three populations of cells: the NY-ESO-1+ TCR+ cells (non-virally replaced TCR) (red), the NY-ESO-1 TCR cells (TCR-knockout) (grey), and the NY-ESO-1 TCR+ cells (those thatprobably retained their native TCR but did not have the NY-ESO-1-specific knock-in TCR) (orange). ), T32 DK007418 (T.L.R. Genome Biol.
Protocol for Electroporation of Cas12a Ribonucleoprotein (RNP - NEB and K.C.H. k, Non-viral TCR replacement was consistently observed at four days after electroporation in both gatedCD8+ and CD4+ T cells across a cohort of six healthy blood donors. For example, mRNA is an effective substrate for inducing rapid protein expression, as it enables results to be achieved more quickly than using DNA. Natl Acad. A.M. is a Chan Zuckerberg Biohub investigator. e, Four days after electroporation of different GFP templates along with a corresponding RNP into primary CD3+ T cells from six healthy donors, GFP expression was observed across both templates and donors. eLife 6, e21856 (2017). The main drawback of the technique is the low transfection efficiency achieved when it is used for primary cell transfection, stem cell transfection and when working with suspension cell lines (mainly due to the fact that it can be cytotoxic and relies on cell division for success). Corn and S. Pyle for suggestions and assistance.
An Efficient Low Cost Method for Gene Transfer to T Lymphocytes Four days after electroporation of the two indicated HDR templates along with their two respective on-target RNPs, the percentage of cells positive for each template was analysed by gating on cells either positive or negative for the other template. Bioz Stars score: 86/100, based on 1 PubMed citations. e, HDR template for endogenous TCR replacement at the TCR- locus. Nature 540, 144149 (2016).
a, Long ssDNA templates have potential to reduce homology-independent integrations while preserving on-target efficiency. Transient electroporation of chemically synthesized, preprocessed, short siRNA duplexes directly into the cell lines of interest may be faster and lead to more efficient mRNA and protein knockdown, especially for hard-to-transfect cell lines. Modalities You can use it to deliver a wide range of substrates directly to the nucleus into your cells, including substrates as DNA, mRNA, miRNA and siRNA. Homology-directed recombination for enhanced engineering of chimeric antigen receptor T cells. By 144h, T cell to cancer cell ratios of less than 1:16 showed evidence of robust target cell killing. In short, transient transfection exerts a temporary influence on your cells, whereas stable transfection leads to permanent genetic changes that are usually passed on to future cell progeny. Article On the plus side, you can use it to transfect large DNA fragments and achieve good transfection efficiencies using cell lines. f, Although these CD3+CD4+CD127loCD45RO+TIGIT+ potential Treg cells were highly enriched for FOXP3 and showed some suppressive capacity when cultured with CFSE-labelled stimulated Tresp cells from healthy donors, CD3+CD4+CD127loCD45RO+TIGIT+ from the compound heterozygotesdid not show suppressive ability. For more than 20 years, Nucleofector Technology has led the market as the most effective non-viral cell transfection method, which can be used even for hard-to-transfect cells, such as primary cells and pluripotent stem cells.
Optimization of the transfection of human THP-1 macrophages by - PubMed Taken together, these data suggest that pre-incubation of the RNP and HDR template, even for a short period, increased the amount of DNA HDR template delivered into the cell, which increased efficiency but decreased viability. and C.G. c, CD4+ T cells isolated by FACS showed detectable GFP+ cells indicative of efficient editing, albeit at lower rates than targeting in CD4+ cells isolated by negative selection (potentially due to the added cellular stress of sorting). How can you choose the best transfection method for your cells? As the method requires successful DNA integration into the host genome, it is often much harder to achieve than transient transfection, and typically has lower transfection efficiency. To successfully perform this technique, you usually need to test and optimize the duration and strength of the pulses against your specific cell type and construct. Most commercial electroporation systems designed for transfection of primary mammalian cells (e.g., Lonza Nucleofector 4D, ThermoFisher Neon) use cuvettes for bulk, batch processing. Open Access articles citing this article. Here, long ssDNA HDR templates with D10A nickase showed lower efficiencies of GFP integration at the RAB11A site. 4h), potentially due to knockout of the endogenous TCR, endogenous regulation of expression of the new TCR, some difference in the cell populations amenable to non-viral versus lentiviral editing, or confounding variables in cell handling between lentiviral transduction and non-viral genome targeting. A.R. PubMed However, transfection efficiency in primary cells is low, mostly due to the high mortality rates caused by the electric pulses. It is also possible to transfect cells with regulatory or non-coding RNAs to modify the expression of endogenous genes (e.g. Sorted NY-ESO-1 TCR+ T cells (3106) generated either by lentiviral transduction (black) or non-viral TCR replacement (red) were transferred on day 7 after tumour seeding and compared to vehicle-only injections until 24days after tumour seeding. discussed the use of ssDNA. The total percentage of cells with bi-allelic HDR integrations must be the sum of genotypes D, E and F. Although the proportion of cells with genotype E (dual fluor positives) is immediately apparent from the phenotypes, genotypes D and F are not. j, Functional cytokine production was observed selectively after antigen exposure in gated CD4+ T cells, similarly to gated CD8+ T cells (Fig. Leonetti, M. D., Sekine, S., Kamiyama, D., Weissman, J. S. & Huang, B. 3 Bi-allelic and multiplexed non-viral genome targeting.
Efficient homology-directed gene editing by CRISPR/Cas9 in - Nature Provided by the Springer Nature SharedIt content-sharing initiative. d, Similarly, increased proportions of IL-2R+FOXP3+ cells are seen 9days after electroporation in the HDR correction conditions in compound heterozygote patients. Extended Data Fig. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells. Browse: Cell and Transfection, Citation, FAQ, Culture Media or use the search below: Filter by Category, Research Area and more Email The Americas: scientific.support@lonza.com Europe and International: scientific.support.eu@lonza.com Phone The Americas: +1 800 521 0390 Monday-Thursday 8:00am-5:30pm EST This construct could express GFP at off-target integration sites, which allowed us to assay for off-target events at the single-cell level using flow cytometry. developed the exonuclease-based ssDNA production method. Examples of such applications include short-term gene expression studies, gene knockdown studies and small-scale protein production. We have established an electroporation protocol for transfection of premature adherent human THP-1 macrophages using Lonza Nucleofector technology. g, GFP integration efficiencies as presented in f but graphed on a logarithmic scale reveal lower levels of functional off-target integrations when using the D10A nickase compared to spCas9 (with an individual off-target gRNA, targeting CXCR4), probably due to the requirement for the D10A nickase to have two gRNAs bound in close proximity to induce a dsDNA break. CAS
Efficient, high-throughput transfection of human embryonic stem cells is co-founder and a member of the scientific advisory board of PACT Pharma.
News | Lonza Sharfe, N., Dadi, H. K., Shahar, M. & Roifman, C. M. Human immune disorder arising from mutation of the alpha chain of the interleukin-2 receptor. Google Scholar. Oncol. ADS PubMed This reduced IL-2R expression could be partially rescued by inclusion of an ssODN HDR template (green) and even more substantially rescued using a large dsDNA HDR template (yellow). c, Diagram of HDR-mediated insertions at the N terminus of a target locus. When transfecting plasmid DNA, also the plasmid backbone and the promoter can have an influence on the transfection result. You've come to the right place! Not every off-target integration will yield fluorescent protein expression (for example, only part of the template sequence could be integrated or it could be integrated in a way that does not lead to measurable expression), but the relative differences in functional off-target expression between different templates and editing conditions can be assayed. h, i, TCR replacement by targeting an entire new TCR into TRAC (ad, also possible with a multiplexed knockout of TCRB), an entire new TCR into TRBC1/2 (f), or multiplexed replacement with a new TCR- into TRAC and a new TCR- into TRBC1/2. For d, e and h, one representative donor is shown. We constructed a model to account for bi-allelic integrations of the same fluorescent protein (Supplementary Note1). and A.M.; US patent application no. By submitting a comment you agree to abide by our Terms and Community Guidelines. 4b), NY-ESO-1+ non-virally edited T cells preferentially accumulated in the tumour versus the spleen. n=5 mice for each of four human T cell donors. Notably, the RNP alone resulted in IL-2R surface expression in almost 50% of edited T cells in all three compound heterozygotes. For example, to create induced pluripotent stem cells (iPSCs) using mRNA, you must deliver it repeatedly over time. Viral transfection (also called viral transduction) takes advantage of the natural ability of certain viruses to carry foreign genes into host cells. Compared to a cohort of 12 similarly edited healthy donors (Fig. Contrary to expectations, we found that co-electroporation of human primary T cells with CRISPR-Cas9 ribonucleoprotein .