In+vivo+expression+technology

=In vivo expression technology=

**In vivo expression technology** (also **IVET**) is a method used to determine which genes are upregulated under particular environmental conditions //in vivo//; especially which bacterial genes are upregulated either to promote infectivity or during the course of infection.

The procedure involves partially digesting genomic DNA from the pathogen of interest to liberate multiple promoter-containing gene fragments, and then ligating the promoter-containing genes into plasmids, upstream of a promoter-less reporter gene, such as one for antibiotic resistance. The plasmid should also contain a gene which confers mobilisation into the pathogen.

The recombinant plasmid is transformed into //E. coli.// The gene for specific mobilisation ensures that the plasmid is conjugated from //E. coli// to the pathogen of interest; and the foreign promoter-containing gene ligated within the plasmid permits homologous recombination between the plasmid and the pathogen's chromosome. Consequently, a series of pathogen recombinants are generated, where reporter genes are fused randomly to different promoter-containing genes from the pathogen.

The recombinant pathogens are used to inoculate a host, such as a mouse. Those promoters whose activity is upregulated during infection will have active antibiotic-resistance genes, while those who activity is not upregulated will die in the presence of antibiotics. The pathogens who survive are isolated, have their plasmids removed and the respective promoter-containing fragment sequenced to identify the gene.


 * An example using the pathogen //V. cholerae// **

Perform a partial digest of //V. cholerae// genomic DNA to liberate promoter-containing gene fragments, and ligate the fragments into a pIVET plasmid, upstream of a promoter-less resolvase gene; the normal function of resolvase is to excise the gene for tetracycline resistance. If the promoter of this gene is up-regulated during infectivity, then these bacteria will //lose// tetracycline resistance.

Inoculate mice with the recombinant bacteria, and recover those who are tetracycline-sensitive. Isolate the plasmids from these strains and sequence the ligated fragment to ascertain which gene sequences are up-regulated during the course of infection.