According the Clontech, "The MSCV Retroviral Expression System (murine stem sell virus) contains vectors that are optimized for introducing and expressing target genes in pluripotent cell lines, including murine or human hematopoietic, embryonic stem (ES), and embryonal carcinoma (EC) cells. They can also be used effectively with most mammalian cell lines".
Considering B cells belong to the hematopoietic lineage, I tried transfecting MSCV-GOI (with MESV psi promoter) into human B-LCLs. Does anyone know why it did not work? Is it because of the promoter incompatibility? I will really appreciate any suugestions/clarifications regarding the underlying mechanism. Thanks.
We tried with no success, the we stably infected with Lent-EF1A-CAR-PGK-Puro and selected with puromycine followed by adenoviral infection which worked well for us. Lentivirus works but still less efficient still was enough to stably express CAR
Srishti Chakravorty , Were you able to transduce your BLCLs with retroviral? What did you do if it did not work? Thanks
Jackkelly
We were unable to infect with MSCV properly. we used lenti method with puro selection. In case you need very quick screening then I would recommend lenti based CAR receptor expression followed by adenoviral infection which worked well above >80% many B cell lymphoma cell lines
Kumar Dilip Thanks for the info.
Can I get the vector construct info and packaging cell line you used?
Jack I used 2nd gen EF1A-DEST- PGK-PURO. I CLONED CAR THROUGH LR GATEWAYI USED Xfext, Lentix packeging CLONTECH with GAG-POL WITH VSVG
Jack Kelly We were not able to transduce these cells with MSCV. One possible reason could be that the promoter is murine specific so won't be transcribed efficiently in a mammalian cell (which we were using). We ended up nucleofecting these cells using the Lonza nucleofector.
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