I want to insert GFP into my lentiCRISPRv2 backbone so that I can immediately see transfection efficiency and lentiviral infected cells instead of waiting for purokill. Does anyone have experience with this? Any tips on which restriction site I should use to insert the GFP?
It isn't the easiest of clonings. I have to check better at the map but at first glance there are not unique restriction enzymes to excise puro-r and replace it for egfp. Addgene should provide a vector with similar characteristics but with eGFP. The best option would be directly yo purchase such vector.
If you want to modify theplasmid you have instead, yiu should consider heavy plasmid reconstruction. Also taking into account that puro is linked to cas9 through a self-cleaving peptide that must be kept in frame with cas9. The easiest thing would be exicsing all cas9::T2a::puro and cloning a new chimeric insert by using gibson assembly. I can provide more details on this and I am used to modify plasmids.for my own needs. However in this case, as I said, I would just search for another vector.
Hope it helps
Francesco
It is true, it's difficult. But as BamH1 is present just before P2A-PuroR*. And Mlu1 RE sites present twice in LentiCRISPRv2 (1st at 231 and 2nd just after P2A-PuroR* 9950). (Read * as stop codon). So you can choose 2 strategies: First you can easily find P2A-eGFP* or T2A-eGFP* template from any other plasmid and amplify with PCR using forward primer with BamH1 and reverse primer with BstE2 RE. Digest vector and PCR with BamH1 and BstE2. Ligate digested vector and PCR product. Since stop codon is present before partially digested PuroR and eGFP, so you are good with this.
Second you can amplify P2A-eGFP* template from any other plasmid and amplify with PCR using forward primer with BamH1 and reverse primer with Mlu1 RE. Digest amplified PCR with Mlu1 and BamH1. Also digest LentiCRISPRv2 with Mlu1 and BamH1. This will give you 3 bands (663bp, 5154bp and 9056bp). Elute the largest band (~9056bp) as well as second large band (5154bp). Treat second large band (5154bp) with Calf intestinal phosphatase (CIP) and Purify with column. And now ligate all three together (P2A-eGFP*, largest and second large band of LentiCRISPRv2). This construction require the screening of colonies with appropriate direction as second large band can ligate in either direction with Mlu1. Hope this can work for you....
Best,
Manoj
You can also clone your gRNA in the pLentiCRISPR-GFP plasmid available from Addgene. That's more simple I guess.
- Badges
- Science method