Nanofountain Probe Electroporation for Monoclonal Cell Line Generation

Espinosa, Horacio D.; Mukherjee, Prithvijit; Patino, Cesar A.

doi:10.1007/978-1-4939-9740-4_6

Cited by 2 publications

(2 citation statements)

References 14 publications

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“…A hollow pyramidal electrode, with an aperture of 500 nm in diameter, which was filled with electrolyte and macro-molecules to be delivered, was controlled by a closed-loop micromanipulator to downwardly approach the upper cell membrane. Then, a short electric pulse was applied across the cell membrane to create a nanopore to let the macro-molecules transfer into the cell before it resealed [108][109][110]. Another case, the nanostraw system, which was built on vertical alumina nanostraws extending from a horizontal track-etched cell culture membrane, formed an array of hollow nanowires connected to an underlying microfluidic channel.…”

Section: Forms and Delivery Methods Of Programmable Site-specific Endonucleases/dna Editorsmentioning

confidence: 99%

Points of View on the Tools for Genome/Gene Editing

Chuang

Lin

2021

IJMS

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Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H840A) and RuvC nuclease activity null mutant (D10A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D10A), Cas9(H840A), and Cas9(D10A, H840A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.

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Section: Forms and Delivery Methods Of Programmable Site-specific Endonucleases/dna Editorsmentioning

confidence: 99%

Points of View on the Tools for Genome/Gene Editing

Chuang

Lin

2021

IJMS

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“…Recent advances in the fields of microfluidics and biosensors have led to transformative capabilities for cell analysis and manipulation with significant implications in cell engineering and fundamental cell biology research . Specifically, mechanisms to open reversible pores in the cell membrane were developed to enable the delivery of exogenous materials into live cells, targeting a broad range of applications from gene editing and cell-line generation to stem-cell reprogramming. − Physical membrane-poration methods, i.e., electroporation , and mechanoporation, − provide enhanced dosage control and uniformity across various cell types compared to lipid–vesicle carriers and viral vectors . Furthermore, improvements to both dosage control and cell health are possible through the miniaturization of these technologies down to the micro- and nanoscales. ,, A nanoscale method known as localized electroporation, utilizes geometric confinements, i.e., nanochannels, to localize an applied electric field to a small area fraction on the cell membrane that results in the formation of pores when the electric potential across the cell membrane (transmembrane potential or TMP) exceeds a threshold value (∼0.5 V). ,− The localized electric field enables the formation of pores at low applied voltages, compared to bulk electroporation systems, and facilitates electrophoretic transport of charged molecules into the cytoplasm. ,− Consequently, the amount of delivered material in these systems can be tuned by adjusting the electrical stimulation parameters such as applied voltage and pulse duration while maintaining high cell viability necessary for repeated stimulations.…”

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confidence: 99%

High-Throughput Microfluidics Platform for Intracellular Delivery and Sampling of Biomolecules from Live Cells

et al. 2022

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Nondestructive cell membrane permeabilization systems enable the intracellular delivery of exogenous biomolecules for cell engineering tasks as well as the temporal sampling of cytosolic contents from live cells for the analysis of dynamic processes. Here, we report a microwell array format live-cell analysis device (LCAD) that can perform localized-electroporation induced membrane permeabilization, for cellular delivery or sampling, and directly interfaces with surface-based biosensors for analyzing the extracted contents. We demonstrate the capabilities of the LCAD via an automated high-throughput workflow for multimodal analysis of live-cell dynamics, consisting of quantitative measurements of enzyme activity using self-assembled monolayers for MALDI mass spectrometry (SAMDI) and deep-learning enhanced imaging and analysis. By combining a fabrication protocol that enables robust assembly and operation of multilayer devices with embedded gold electrodes and an automated imaging workflow, we successfully deliver functional molecules (plasmid and siRNA) into live cells at multiple time-points and track their effect on gene expression and cell morphology temporally. Furthermore, we report sampling performance enhancements, achieving saturation levels of protein tyrosine phosphatase activity measured from as few as 60 cells, and demonstrate control over the amount of sampled contents by optimization of electroporation parameters using a lumped model. Lastly, we investigate the implications of cell morphology on electroporation-induced sampling of fluorescent molecules using a deep-learning enhanced image analysis workflow.

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Nanofountain Probe Electroporation for Monoclonal Cell Line Generation

Cited by 2 publications

References 14 publications

Points of View on the Tools for Genome/Gene Editing

Points of View on the Tools for Genome/Gene Editing

High-Throughput Microfluidics Platform for Intracellular Delivery and Sampling of Biomolecules from Live Cells

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