Next, we tested whether ORANGE knock-ins could also be delivered using lentiviral (LV) vectors. We divided the ORANGE knock-in cassette over two LV constructs (S4A Fig) because the full cassette exceeds the packaging limit of LV particles. Also, premature coexpression of Cas9 and the gRNA during the production of viral particles in packaging cells would lead to removal of the donor DNA. Both in dissociated hippocampal cultures and in organotypic slice cultures, we observed knock-ins, showing that LVs can be used to successfully express ORANGE knock-ins (S4 Fig). Together, these results show that ORANGE is compatible with various modes of DNA delivery suitable for labeling in dissociated neuronal cultures, in organotypic slice cultures and in vivo, broadening the potential applications of this CRISPR/Cas9 genome editing toolbox.
recovery toolbox for illustrator full 31
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