PQCXIX and mCherry for murine T cell transduction any protocol?
Hi Nik,
1. pQCXIX possesses a CMV promoter, which will be inactivated in murine T cells. You need to switch to a vector using other promoters, like MSCV, PGK or EF1. I have used the pMIG and pMIG-II retroviral vector to transduce murine T cells and bone marrow cells. And the transduction efficiency reached as high as 90% using a MOI of 10. Keep in mind that you need to pre-activate murine T cell before retroviral infection. E.g, plate-bound aCD3/28 stimulation, 5ug/ml, overnight. If you do not pre-activate murine T cells, very few retrovirus can integrate into the genome of murine T cells, which means although the retrovirus can only get into the cells, but many of them would fail to integrate into the cellular genome to express your cDNA.
2. Another issue is that mCherry is not as good as GFP or Ametrine. In my hand, when using the same MOI of two retroviral vectors with the same backbone (e.g. the only difference of pMSCV-IRES-mCherry and pMSCV-IRES-GFP-II is their fluorescent protein) to transduce activated murine T cells, the mCherry vector consistently show much lower transduction efficiency. E.g., 40% vs. 90%. pMIG-II is a vector you can get from Addgene.
Hope you will get it to work.
Best,
Lingyun
Hi Lingyun !
Could you share which packing and coat plasmids shall I use for making virus using HEK 293T cells.
Thanks !
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