Working with CRISPR-Cas editing in suspension cells. I need a successful delivery of plasmids to suspension cells using nucleofection method ( Lonza Amaxa 4D-X system). The problem is this nucleofection system/technology is working well with small size plasmid ( 3kb-pmaxGFP) but not large size plasmids ( 10 kb). I have tried the following programs for my cell line ( B cells) and used 3 kb GFP plasmids: CA-137, CM-138, CM-137, CM-150, DN-100,DS-138,DS-137,DS-130, DS-150,DS-120,EH-100,EO-100,EN-138,EN-150 and EX-113. I have used SF buffer and also suggested home made buffers in publications ( Article An Efficient Electroporation Protocol for the Genetic Modifi...
). Does any of you use nucleofection to deliver Crispr-Cas or any large size plasmids to suspension cells? What is expected transfection efficiency for a good editing efficiency. I transfect million cells per transfection in 100 uL buffer. I co-transfect 750 ng large size plasmid with 1500 ng 3 kb size plasmid. I also take into consideration the following paper which nicely showed co-transfecting with small plasmids facilitate the large plasmid entry to cells (Article Successful delivery of large-size CRISPR/Cas9 vectors in har...
). Any suggestion, advice, comment is appreciated! Thanks!Questions?
1- Does nucleofection work efficiently to deliver big plasmids to suspension cells?
2- Do you have experience with Lonza 4D-X programes on B cells?
3- What is the best ratio when doing co-transfection CRISPR-Cas9: gRNAs?
4- What buffers work well when transfecting suspension cells?
Hello, small vectors can rapidly pass cell and nuclear membranes and large vectors can move on this flow of small vectors into the cell. By co-travelling with small vectors, larger vectors may enter the cell without getting entangled in two or more open membrane pores, which ensures proper plasmid uptake and membrane reclosure thereby preventing cell death. Unlike transcribing the CRISPR/Cas9 system inside the host cell a recombinant CRISPR/Cas9 ribonucleoprotein can be formed in vitro prior to electroporation. This approach yielded higher transfection efficiencies.
See attached info may be useful for you.
Hi Sibel,
I also work with B cells and electroporation of big plasmids (approx. 10 kbp) by using Amaxa Nucleofector I (Lonza) along with the Cell Line Nucleofector Kit V. When transfecting the pmaxGFP, the transfection efficiency is around 60-70%, whereas it is reduced to about 10% when transfecting with the 10 kbp plasmids. I use the X-001 programme. Such efficiency might be sufficient for further applications, especially if you are selecting your cells with a cell sorter based on GFP expression. What is your transfection efficiency?
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