Primary T cell electroporation. RNPs and HDR templates were electroporated 2 days after initial T cell stimulation. T cells were collected from their culture vessels and magnetic anti-CD3/anti-CD28 dynabeads were removed by placing cells on an EasySep cell separation magnet for 2 min. Immediately before electroporation, de-beaded cells were centrifuged for 10 min at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 µl buffer per 1 million cells. For optimal editing, 1 million T cells were electroporated per well using a Lonza 4D 96-well electroporation system with pulse code EH115. Alternate cell concentrations from 200,000 up to 2 million cells per well resulted in lower transformation efficiencies. Alternate electroporation buffers were used as indicated, but had different optimal pulse settings (EO155 for OMEM buffer). Unless otherwise indicated, 2.5 µl RNPs (50 pmol total) were electroporated, along with 2 µl HDR Template at 2 µg µl-1 (4 µg HDR template total).
Highly cited Nature research article:
Swift Gene-Editing Method May Revolutionize Treatments for Cancer and Infectious Diseases.
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