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.
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Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278
Nakagawa M et al (2007) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26:101
Kim D et al (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4(6):472–476
Lai T, Yang Y, Ng S (2013) Advances in mammalian cell line development technologies for recombinant protein production. Pharmaceuticals 6(5):579–603
Büssow K (2015) Stable mammalian producer cell lines for structural biology. Curr Opin Struct Biol 32:81–90
Kim TK, Eberwine JH (2010) Mammalian cell transfection: the present and the future. Anal Bioanal Chem 397(8):3173–3178
Boukany PE et al (2011) Nanochannel electroporation delivers precise amounts of biomolecules into living cells. Nat Nanotechnol 6(11):747–754
Sharei A et al (2013) A vector-free microfluidic platform for intracellular delivery. Proc Natl Acad Sci
Loh OY et al (2008) Electric field-induced direct delivery of proteins by a nanofountain probe. Proc Natl Acad Sci U S A 105(43):16438–16443
Ruiguo Y et al (2018) Monoclonal cell line generation and CRISPR/Cas9 manipulation via single-cell electroporation. Small 14(12):1702495
Mukherjee P et al (2018) A combined numerical and experimental investigation of localized electroporation-based cell transfection and sampling. ACS Nano
Kang W et al (2013) Nanofountain probe electroporation (NFP-E) of single cells. Nano Lett
Kang W et al (2014) Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons. Lab Chip 14(23):4486–4495
Kaufmann T, Ravoo BJ (2010) Stamps, inks and substrates: polymers in microcontact printing. Polym Chem 1(4):371–387
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Espinosa, H.D., Mukherjee, P., Patino, C. (2020). Nanofountain Probe Electroporation for Monoclonal Cell Line Generation. In: Li, S., Chang, L., Teissie, J. (eds) Electroporation Protocols. Methods in Molecular Biology, vol 2050. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9740-4_6
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DOI: https://doi.org/10.1007/978-1-4939-9740-4_6
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9739-8
Online ISBN: 978-1-4939-9740-4
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