in vivo cloning

Recombineering with dsDNA can be used for in vivo cloning by gap repair (Figure 2). Again, a linear substrate is made by PCR but now the DNA fragment contains a plasmid replication origin. The ends of the fragment have homology flanking the region of DNA to be cloned, be it a gene from the chromosome or several introns of a human gene contained on a BAC.

Figure 2. Using dsDNA to in vivo clone by gap repair. In this case, the linear substrate made by PCR contains an origin of replication and a drug-resistance marker as well as 50 bases of homology to the region to be cloned. In this case, three genes are being cloned onto a plasmid from the chromosome or a BAC. If one of the genes were a selectable marker, the DrugR marker need not be on the linear fragment.



The linear plasmid fragment is introduced by transformation into a recombineering-proficient cell and drug resistance is selected. Plasmid DNA is then isolated and clones screened for the correct insert. In contrast with classical genetic engineering, cloning by retrieval avoids mutations in the cloned gene that arise during PCR, making this technique useful for cloning mutant genes for sequencing. Although this type of recombineering is less efficient than gene replacement, with a frequency around 102/108 viable cells for the λ Red system, it is a very useful cloning technique. 

Important note: In 2012, Fu et al. demonstrated that RecET from the Rac prophage is a more efficient system for performing this recombination reaction. In vivo cloing with the RecET system requires the full length RecE protein rather than an N-terminal truncation. 


 © RECOMBINEERING 2016