Chapter 6
Nanofountain Probe Electroporation for Monoclonal Cell
Line Generation
Horacio D. Espinosa, Prithvijit Mukherjee, and Cesar Patino
Abstract
In the field of genetic engineering, the modification of genes to produce stable cell lines has a variety of
applications ranging from the development of novel therapeutics to patient specific treatments. To successfully generate a cell line, the gene of interest must be delivered into the cell and integrated into the genome.
The efficiency of cell line generation systems therefore depends on the efficiency of delivery of genetically
modifying molecules such as plasmids and CRISPR/CAS9 complexes. In this work, we describe a localized
electroporation-based system to generate stable monoclonal cell lines. By employing the nanofountain
probe electroporation (NFP-E) system, single cells in patterned cultures are selectively transfected with
plasmids, grown, and harvested to obtain stably expressing cell lines. Methods for microcontact printing,
cell culture, electroporation, and harvesting are detailed in this chapter.
Key words Genetic engineering, Localized electroporation, Nanofountain probe electroporation,
Plasmid transfection, Monoclonal cell lines, Cell line generation
1 Introduction
Advances in gene editing and cell line generation technologies have
profound implications in healthcare and fundamental biological
research [1–4]. In cell line engineering, modification of the cell’s
genome is accomplished through a variety of methods such as;
plasmid transfection, lentiviral transduction, recombinasemediated cassette exchange (RMCE), and CRISPR/CAS9 gene
editing [1, 5]. Successful gene editing involves the stable expression
of the gene of interest over time. Furthermore, marker genes are
introduced with the gene of interest to distinguish and isolate
successfully edited cells from nonedited cells. For this purpose,
fluorescent markers such as GFP as well as antibiotic resistant
genes are often used [5]. Once the stably edited cells are isolated,
they can be cultured and harvested to form monoclonal cell lines.
Shulin Li et al. (eds.), Electroporation Protocols: Microorganism, Mammalian System, and Nanodevice,
Methods in Molecular Biology, vol. 2050, https://doi.org/10.1007/978-1-4939-9740-4_6,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
59
Nanofountain Probe Electroporation for Monoclonal Cell
Line Generation
Horacio D. Espinosa, Prithvijit Mukherjee, and Cesar Patino
Abstract
In the field of genetic engineering, the modification of genes to produce stable cell lines has a variety of
applications ranging from the development of novel therapeutics to patient specific treatments. To successfully generate a cell line, the gene of interest must be delivered into the cell and integrated into the genome.
The efficiency of cell line generation systems therefore depends on the efficiency of delivery of genetically
modifying molecules such as plasmids and CRISPR/CAS9 complexes. In this work, we describe a localized
electroporation-based system to generate stable monoclonal cell lines. By employing the nanofountain
probe electroporation (NFP-E) system, single cells in patterned cultures are selectively transfected with
plasmids, grown, and harvested to obtain stably expressing cell lines. Methods for microcontact printing,
cell culture, electroporation, and harvesting are detailed in this chapter.
Key words Genetic engineering, Localized electroporation, Nanofountain probe electroporation,
Plasmid transfection, Monoclonal cell lines, Cell line generation
1 Introduction
Advances in gene editing and cell line generation technologies have
profound implications in healthcare and fundamental biological
research [1–4]. In cell line engineering, modification of the cell’s
genome is accomplished through a variety of methods such as;
plasmid transfection, lentiviral transduction, recombinasemediated cassette exchange (RMCE), and CRISPR/CAS9 gene
editing [1, 5]. Successful gene editing involves the stable expression
of the gene of interest over time. Furthermore, marker genes are
introduced with the gene of interest to distinguish and isolate
successfully edited cells from nonedited cells. For this purpose,
fluorescent markers such as GFP as well as antibiotic resistant
genes are often used [5]. Once the stably edited cells are isolated,
they can be cultured and harvested to form monoclonal cell lines.
Shulin Li et al. (eds.), Electroporation Protocols: Microorganism, Mammalian System, and Nanodevice,
Methods in Molecular Biology, vol. 2050, https://doi.org/10.1007/978-1-4939-9740-4_6,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
59