Pronuclear-stage embryos were mixed with preassembled Cas9/gRNA RNPs and subjected to electrical pulses to permeabilize the zona pellucida and the cell membrane in a transient manner to allow access of RNP to the nucleus (Fig. 2A ). The NEPA21 electroporator system and a CUY501P1-1.5 electrode were used (NepaGene, Japan) following the provider's instructions and with a final impedance range of 0.18–0.22 kΩ.
Nepa21 electroporator system
Manufactured by Nepa Gene
Sourced in Japan
The NEPA21 electroporator system is a laboratory instrument used for the delivery of genetic material, such as DNA or RNA, into cells through the process of electroporation. The device generates controlled electric pulses that temporarily increase the permeability of cell membranes, allowing the genetic material to enter the cells.
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Lab products found in correlation
2 protocols using nepa21 electroporator system
1
Pronuclear Embryo Electroporation
2
Generation of Integrin-Expressing Cell Lines
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To generate integrin‐α4 expressing cells, HEK293A cells were transfected with 2‐μg ITGA4 or SVEP1‐FLAG plasmid using lipofectamine 2000 (ThermoFisher) and selected using 800‐μg·ml−1 geneticin 48 h post transfection. Cells were diluted to single cell to isolate individual colonies and clones expressing integrin‐α4 or SVEP1‐FLAG were identified using anti‐integrin α4 or anti‐FLAG antibody respectively (Table S2 ). To generate an integrin α9‐GFP stable line HEK293A cells were transfected using the NEPA21 Electroporator system (Nepagene). Cells were transfected with 10‐μg integrin α9‐GFP plasmid in OptimMEM. After 48 h, cells were selected using 500‐μg·ml−1 geneticin. Cells were diluted to single cell to isolate individual clones, with clones expressing integrin α9‐GFP identified by fluorescent microscopy.
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