Modeling and Experimental Validation of DNA Motion in Uniform and Nonuniform DC Electric Fields

David, Regis A.; Jensen, Brian D.; Black, J.A.; Burnett, Sandra H.; Howell, Larry L.

doi:10.1115/1.4002321

Cited by 8 publications

(7 citation statements)

References 24 publications

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“…Later, nanoinjection was extended to somatic cell targets by utilizing an array of silicon etched lances, a design used in this work. Electrostatic principles used to initially determine DNA behavior with the single lance injector (Aten et al 2011 ; David et al 2010 ) have been also applied in LAN, both structurally (Teichert et al 2013 ; Teichert and Jensen 2013 ) and procedurally (Lindstrom et al 2014 ; Sessions et al 2014 ).…”

Section: Discussionmentioning

confidence: 99%

“…A possible explanation for this behavior is that 4.5 mA protocols offers the best balance between effective electrical attraction/release of the DNA during the LAN process, while being a mild stressor in terms of cell viability. Even though the 1.5 mA protocol is milder in terms of cellular stress, a feature seen in electroporation studies to improve cell viability (Canatella et al 2001 ), perhaps the 4.5 mA protocol is better at balancing the cellular stress with effective attraction and release of the DNA, a parameter shown to increase DNA motion when done at higher magnitudes in processes like electrophoresis (David et al 2010 , 2011 , 2012 ).…”

Section: Discussionmentioning

confidence: 99%

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CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection

et al. 2016

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BackgroundCRISPR-Cas9 genome editing and labeling has emerged as an important tool in biologic research, particularly in regards to potential transgenic and gene therapy applications. Delivery of CRISPR-Cas9 plasmids to target cells is typically done by non-viral methods (chemical, physical, and/or electrical), which are limited by low transfection efficiencies or with viral vectors, which are limited by safety and restricted volume size. In this work, a non-viral transfection technology, named lance array nanoinjection (LAN), utilizes a microfabricated silicon chip to physically and electrically deliver genetic material to large numbers of target cells. To demonstrate its utility, we used the CRISPR-Cas9 system to edit the genome of isogenic cells. Two variables related to the LAN process were tested which include the magnitude of current used during plasmid attraction to the silicon lance array (1.5, 4.5, or 6.0 mA) and the number of times cells were injected (one or three times).ResultsResults indicate that most successful genome editing occurred after injecting three times at a current control setting of 4.5 mA, reaching a median level of 93.77 % modification. Furthermore, we found that genome editing using LAN follows a non-linear injection-dose response, meaning samples injected three times had modification rates as high as nearly 12 times analogously treated single injected samples.ConclusionsThese findings demonstrate the LAN’s ability to deliver genetic material to cells and indicate that successful alteration of the genome is influenced by a serial injection method as well as the electrical current settings.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection

et al. 2016

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“…We have previously presented a model describing the repulsion of DNA into a cell and we have validated the model by experiments [4][5][6][7]. The previous work has shown that the motion of DNA due to electrophoresis may be accurately predicted using the model.…”

Section: Introductionmentioning

confidence: 89%

“…Previous work [4,5] describes the model proposed to determine the motion of DNA using nanoinjection (compare with the models found in [22][23][24][25][26][27][28][29][30], which treat only uniform electric fields). Our model has two main parts.…”

Section: Mathematical Modelmentioning

confidence: 99%

Study of design parameters affecting the motion of DNA for nanoinjection

David

Jensen

Black

et al. 2012

J. Micromech. Microeng.

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This paper reports the effects of various parameters on the attraction and repulsion of DNA to and from a silicon lance. An understanding of DNA motion is crucial for a new approach to insert DNA, or other foreign microscopic matter, into a living cell. The approach, called nanoinjection, uses electrical forces to attract and repel the desired substance to a micromachined lance designed to pierce the cell membranes. We have developed mathematical models to predict the trajectory of DNA. The mathematical model allows investigation of the attraction/repulsion process by varying specific parameters. We find that the ground electrode placement, lance orientation and lance penetration significantly affect attraction or repulsion efficiency, while the gap, lance direction, lance tip width, lance tip half-angle and lance tip height do not.

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“…The damaged bond pad was stripped of much of its gold layer compared to the undamaged bond pad to the right of the bond. Electrolysis and the associated damage were avoided by maintaining applied voltages below 1.8 V. electrical current flow [34]. However, above the decomposition voltage, electrolysis occurs, producing hydrogen molecules at the negative electrode and oxygen molecules at the positive electrode.…”

Section: Introductionmentioning

confidence: 99%

Electrostatic Accumulation and Release of DNA Using a Micromachined Lance

Aten

Jensen

Burnett

et al. 2011

J. Microelectromech. Syst.

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This paper investigates the accumulation and release of deoxyribonucleic acid (DNA) relative to a surface micromachined silicon lance. The lance is a critical element of nanoinjection, a proposed approach for injecting foreign DNA into living cells. The quantity of DNA accumulated on the nanoinjector lance and the speed at which it can be moved on and off the lance are essential to the proposed system's function. Prototype nanoinjector lances were fabricated using a multilayer surface micromachining process. DNA stained with the fluorescent dye 4 , 6-diamidino-2-phenylidole dihydrochloride was visualized using fluorescent illumination as the DNA was accumulated on and released from the tips of microelectromechanical systems (MEMS) microlances using a 1.5-V dc source. In 5 min 46 s, the lance accumulated over 32 000 DNA molecules from a dilute DNA solution. The lance then released over 6200 DNA molecules within 6 s. Finally, the nanoinjector lance was used to inject a reporter gene encoding a red fluorescent protein into a mouse embryo, resulting in expression of the gene. The nanoinjector lance represents an important and significant step in the development of a self-contained MEMS-based DNA injection system.

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Modeling and Experimental Validation of DNA Motion in Uniform and Nonuniform DC Electric Fields

Cited by 8 publications

References 24 publications

CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection

CRISPR-Cas9 directed knock-out of a constitutively expressed gene using lance array nanoinjection

Study of design parameters affecting the motion of DNA for nanoinjection

Electrostatic Accumulation and Release of DNA Using a Micromachined Lance

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