In vivo Recombination

Volkov et al. described a hybrid in vitro-in vivo recombination method involving formation of a heteroduplex between two homologous sequences in vitro and transformation into bacterial cells11251. Mismatches present in the heteroduplex are randomly repaired by the host cell, creating recombinant sequences composed of the elements of each parent. This approach may be particularly useful in recombining large genes or entire operons. 

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Figure 4.7. In vivo recombinational cloning in yeast. Two successive PCR reactions are performed. Each set of primers contains 5 flanking sequences that are eventually used for homologous recombination with vector sequences after transformation of yeast with the 2nd PCR fragment and the linearized vector.

Waterhouse, P, Griffiths, A D., Johnson, K S., and Winter, G. (1993) Combinatorial infection and in vivo recombination, a strategy for making large phage antibody repertoires. Nucleic Acids Res 21,2265,2266. 

Tsurushita, N., Fu, H., and Warren, C. (1996) Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries. Gene 172, 59-63. 

Libraries of such size have so far only been generated for antibody repertoires. These are constructed by in vivo recombination of smaller, primary sublibraries generated by PCR (e.g., Sblattero and Bradbury, 2000). This level of sequence diversity may be attainable for other proteins, as long as they consist of multiple independent folding domains that can tolerate the insertion of recombination sites within the linker region. 

Single protein molecule (Gly4Ser)3 linker can shorten DNA insert 700 bp In vivo recombination published, scFv libraries possible in principle... 

In vivo recombination used to create large libraries... 

The success of in vitro selection depends on the level of functional display but also principally on the quality and the diversity of the starting library. The attainable diversity can be significantly increased by taking advantage of site-specific in vivo recombination (see Section 5.4). In this case, the bacterial host must express the appropriate site-specific recombinase. 

Mastrangeli A, Danel C, Rosenfeld MA, Stratford-Perricaudet L, Perricaudet M, Pavirani A, Lecocq JP, Crystal RG. Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer. J Clin Invest 1993, 91, 225-234. 

FLP recombinase In vivo recombination efifirienry at elevated temperatures in E.coli and mammalian cells in vitro thermostability Improved recombination efficiency in E. coli and mammalian cells... 

In vivo recombination of homologous genes + screening E. coli [122]... 

Random mutagenesis + in vivo recombination + site-directed mutants + screening Yeast [123]... 

Shao, Z., Kratzsch, P., Schmuck, R., von der Eltz, H., Kenklies, J. (2003) AWalkThrough technique for in vitro/in vivo recombination of polynucleotide sequences. EP1404825. 

In-vivo recombination Methodologically more difficult. Can produce unwanted by products. Will produce the highest diversity with the least work, as infection rather than transfection is used. 

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