I want to use the Cre LoxP to knock out my target gene in a mammalian cell line. According to the mechanism, I need to add two loxP sequences at both ends of the gene through homologous recombination. I haven't done this before in eucaryptes, but I did homologous recombination in E. coli with λ-red. My question is for the Cre lox system, does Cre have the same function as λ-red? If yes, my plan is to transfect the cre-containing plasmid into the cell line first to obtain a cre-forming cell line. (How to activate cre within the cell?) Secondly, design the primers composed of 20bp of my target gene (from start codon) followed by the 34bp loxP sequence and 20bp of a drug-resistant gene on a plasmid. Then use the primers to amplify the plasmid and transfect my cre cell line with the PCR product. Will cre recombine the lox-drug R-lox part to the genome to replace my target gene first and then do the deletion through lox? If no, what recombinase can I use or is something wrong with my plan? Thank you.
No it does not. Cre is a recombinase (like ΦC31 integrase) while the Lambda red system consists of an exonuclease, single strand annealing protein and NHEJ inhibitor. The mechanisms are completely different with transposases cutting, becoming covalently attached, pivoting to cause strand exchange and then rejoining the backbone. The lambda red system is analogous to the RAD52 homologous repair pathway when with a double strand break an exonuclease cuts the one strand to form a single stranded overhang which is then used by the single strand annealing protein to perform strand invasion which is then repaired by DNA polymerases.
Conditional Cre expression is usually done using a chemically inducible promoter like Tet-On systems. For homologous recombination with eukaryotes you need longer homology arms of 500-3000bp.
Your experimental design needs to be rethought. You need two rounds of homologous recombination to add lox sites upstream and downstream of your target CDS using homology arms for the promoter and start of the CDS and end of the CDS and some downstream sequence. You then co-transfect your plasmid with the selection marker and the Cre expressing plasmid which will then perform a cassette exchange.
If you want an analogous system to Lambda red for eukaryotes you will need to use ICP8 from Herpes simpex.
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