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Review
. 2002 Dec;70(12):6518-23.
doi: 10.1128/IAI.70.12.6518-6523.2002.

In vivo expression technology

Michael J Angelichio  1 , Andrew Camilli
Affiliations
Review

In vivo expression technology

Michael J Angelichio et al. Infect Immun. 2002 Dec.
No abstract available

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Figures

FIG. 1.
FIG. 1.
Graphic depiction of four variations of IVET. Auxotrophy complementation-based selections are conducted using fusions to a promoterless purA or other such gene (plasmid 1), antibiotic selections are conducted using fusions to a promoterless antibiotic gene such as cat (reporter 2), dual reporter selections can use reporter genes such as hly (reporter 3) or other types of genes that provide an in vivo selection and an in vitro screen for promoter activity, and finally, RIVET screening is done using a promoterless tnpR gene (reporter 4), whose protein product will excise a substrate cassette (res1-tet-res1 in figure) from elsewhere in the bacterial genome. Reporter gene fusion libraries are constructed by ligating random genomic fragments (designated gene X′) into the IVET vector of choice, followed by transformation into the pathogen of interest. The suicide plasmids then recombine into the chromosome by insertion-duplication, creating a merodiploid. In the case of RIVET, a prescreen is required to remove strains harboring gene fusions that are active in vitro: this is accomplished by selecting for tetracycline-resistant, LacZ− colonies. In all cases, fusion strains are passaged through an appropriate animal model of disease and collected from infected tissues or fluids after a period of time. In the case of the antibiotic-based IVET, the antibiotic (in this example, chloramphenicol) must be present at sufficient concentrations in animal tissues to select for in vivo expression of the gene fusion. Strains containing infection-induced gene fusions to purA and cat are selected in the host and are subsequently screened for lack of in vitro expression on LacZ indicator plates. Alternatively, L. monocytogenes strains containing infection-induced gene fusions to hly are selected in the host (see text for details) and are subsequently screened for lack of in vitro expression on blood agar plates (Hly will lyse the red blood cells, forming a zone of clearing around colonies). Finally, infection-induced gene fusions to tnpR are screened for at a postinfection stage by virtue of their tetracycline sensitivity and lack of expression of LacZ on indicator plates.
See this image and copyright information in PMC

References

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