Dear All,
I would like to use these two plasmids from Addgene. How can I know these plasmids are 2nd or 3rd generation? In addition, these plasmids don't have 5'LTR. In fact, two 3'LTRs are presented. Can the gene flanking by these two 3'LTR be expressed after transduction.
Moreover, can I use these two lentiviral transfer vectors for transient transfections into fibroblast?
Addgene #60903: pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP
Addgene #79372: pHRdSV40_scFv_GCN4_sfGFP_p65-hsf1_GB1_NLS Sequences
Salah A. Alshehade
- They are likely 3rd generation vectors. Key features of 3rd gen vectors are the split packaging system (vectors lack viral genes) and additional safety modifications like self-inactivating (SIN) LTRs. In SIN LTRs, the promoter/enhancer region is deleted from the 3' LTR, making it transcriptionally inactive after integration. This increases safety.
- Yes, the transgene flanked by the two 3' LTRs should be expressed after transduction. The internal promoter (like CMV or SV40) will drive expression of the cassette.
- These transfer vectors cannot replicate on their own without packaging into viruses, so they are not suitable for transient transfection alone. To express the transgenes in fibroblasts transiently, you would need to clone them into a standard expression plasmid.
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