The overall efficiency of stable cell line generation depends on
both the efficiency of delivery of gene editing molecules into the
cell as well as the efficiency of integration of the DNA of interest in
the genome. The focus of this protocol is to enhance the efficiency
of delivery. Lipid-mediated delivery methods are inefficient, celldependent, and lack dosage control [6]. On the other hand, viral
transduction protocols have higher delivery efficiencies but pose
biohazard safety issues [5]. As an alternative, physical delivery
systems like electroporation and mechanoporation can be
employed to deliver precise doses of biomolecules into cells without
incurring the biosafety hazards of viral vectors [7, 8]. In bulk
electroporation systems, cells in suspension are subjected to an
electric field that induces pore formation across the cell membrane.
However, the viability of cells in bulk electroporation systems is low
due to the strong electric fields the cells are subjected to. Localizedelectroporation systems circumvent this issue by confining the
electric field to small areas on the cell membrane, thus resulting in
high cell viability [7, 9], uniform delivery [10], and high delivery
efficiencies [11]. In these methods, the electric field is applied
across nanostructures such as nanopores, nanoprobes, or nanopipettes [7, 9–13]. In this protocol the nanofountain probe electroporation system (NFP-E) is employed to transfect cells grown on a
culture dish patterned with membrane proteins in an array format
[10]. The transfection imparts green fluorescent protein (GFP)
expression and antibiotic (zeocin) resistance to the cells. The stably
transfected cells are then selected via antibiotic treatment and
harvested.
2 Materials
2.1 Mold Fabrication
1. The mold fabrication for the PDMS stamps was performed in a
clean-room facility having the following equipment: spin
coater, hot plates, reactive ion etcher (Samco Inc., Kyoto,
Japan), mask aligner (SUSS Microtec SE, Garching, Germany),
and Parylene coater (SCS, Indianapolis, IN, USA).
2. Si wafers—Wafers are used for the lithographic patterning of
photoresist features.
3. SU-82000 photoresist (MicroChem, Westborough, MA,
USA)—Negative photoresist used for fabricating high aspectratio microscale features.
4. SU-8 Developer (MicroChem)—Developer is used to remove
photoresist material not exposed to UV.
2.2 PDMS Stamp
Fabrication
1. Polydimethylsiloxane (PDMS) Sylgard 184 (Dow Corning,
Midland, MI, USA)—Mixed in a 10:1 elastomer to curing
agent ratio and used for microcontact printing.
60
Horacio D. Espinosa et al.
both the efficiency of delivery of gene editing molecules into the
cell as well as the efficiency of integration of the DNA of interest in
the genome. The focus of this protocol is to enhance the efficiency
of delivery. Lipid-mediated delivery methods are inefficient, celldependent, and lack dosage control [6]. On the other hand, viral
transduction protocols have higher delivery efficiencies but pose
biohazard safety issues [5]. As an alternative, physical delivery
systems like electroporation and mechanoporation can be
employed to deliver precise doses of biomolecules into cells without
incurring the biosafety hazards of viral vectors [7, 8]. In bulk
electroporation systems, cells in suspension are subjected to an
electric field that induces pore formation across the cell membrane.
However, the viability of cells in bulk electroporation systems is low
due to the strong electric fields the cells are subjected to. Localizedelectroporation systems circumvent this issue by confining the
electric field to small areas on the cell membrane, thus resulting in
high cell viability [7, 9], uniform delivery [10], and high delivery
efficiencies [11]. In these methods, the electric field is applied
across nanostructures such as nanopores, nanoprobes, or nanopipettes [7, 9–13]. In this protocol the nanofountain probe electroporation system (NFP-E) is employed to transfect cells grown on a
culture dish patterned with membrane proteins in an array format
[10]. The transfection imparts green fluorescent protein (GFP)
expression and antibiotic (zeocin) resistance to the cells. The stably
transfected cells are then selected via antibiotic treatment and
harvested.
2 Materials
2.1 Mold Fabrication
1. The mold fabrication for the PDMS stamps was performed in a
clean-room facility having the following equipment: spin
coater, hot plates, reactive ion etcher (Samco Inc., Kyoto,
Japan), mask aligner (SUSS Microtec SE, Garching, Germany),
and Parylene coater (SCS, Indianapolis, IN, USA).
2. Si wafers—Wafers are used for the lithographic patterning of
photoresist features.
3. SU-82000 photoresist (MicroChem, Westborough, MA,
USA)—Negative photoresist used for fabricating high aspectratio microscale features.
4. SU-8 Developer (MicroChem)—Developer is used to remove
photoresist material not exposed to UV.
2.2 PDMS Stamp
Fabrication
1. Polydimethylsiloxane (PDMS) Sylgard 184 (Dow Corning,
Midland, MI, USA)—Mixed in a 10:1 elastomer to curing
agent ratio and used for microcontact printing.
60
Horacio D. Espinosa et al.