An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells.
Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza's Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing ofPDCD1gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology.
Programa de Carcinogênese Molecular, Coordenação de Pesquisa, Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil; Fundação Instituto Oswaldo Cruz, Vice-presidência de Pesquisa e Laboratórios de Referência, Rio de Janeiro, Brazil.
Leonardo
L
Chicaybam
Programa de Carcinogênese Molecular, Coordenação de Pesquisa, Instituto Nacional de Câncer (INCA) , Rio de Janeiro , Brazil.
Camila
C
Barcelos
Programa de Carcinogênese Molecular, Coordenação de Pesquisa, Instituto Nacional de Câncer (INCA) , Rio de Janeiro , Brazil.
Barbara
B
Peixoto
Programa de Carcinogênese Molecular, Coordenação de Pesquisa, Instituto Nacional de Câncer (INCA) , Rio de Janeiro , Brazil.
Mayra
M
Carneiro
Programa de Carcinogênese Molecular, Coordenação de Pesquisa, Instituto Nacional de Câncer (INCA) , Rio de Janeiro , Brazil.
Cintia Gomez
CG
Limia
Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Centro de Transplante de Medula Óssea, Instituto Nacional de Câncer, Rio de Janeiro, Brazil.
Patrícia
P
Redondo
Banco de Cordão Umbilical e Placentário, Instituto Nacional de Cancer (INCA), Rio de Janeiro, Brazil; Instituto Fernandes Figueira, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
Carla
C
Lira
Banco de Cordão Umbilical e Placentário, Instituto Nacional de Cancer (INCA) , Rio de Janeiro , Brazil.
Flávio
F
Paraguassú-Braga
Instituto Fernandes Figueira, Fundação Oswaldo Cruz , Rio de Janeiro , Brazil.
Zilton Farias Meira De
ZF
Vasconcelos
Programa de Carcinogênese Molecular, Coordenação de Pesquisa, Instituto Nacional de Câncer (INCA) , Rio de Janeiro , Brazil.
Luciana
L
Barros
Programa de Carcinogênese Molecular, Coordenação de Pesquisa, Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil; Fundação Instituto Oswaldo Cruz, Vice-presidência de Pesquisa e Laboratórios de Referência, Rio de Janeiro, Brazil.
Martin Hernán
MH
Bonamino
CD34; CRISPR; GFP; MSC; PD-1; T lymphocyte; cell line; electroporation; transposon