Monitoring of Biomarker Expression

Biomarkers have become key tools for physiological studies. They enable:

  • Detection of an early event in the response cascade.
  • Quantification of a physiological response.

Knockin models could be designed in order to provide valuable tools for the monitoring of biomarker expression. Following this approach a reporter marker will be expressed concomitantly to the biomarker. To avoid interference with the biomarker biological activities, genOway combines its IRES technology with the Humanization Knockin technology in order to achieve bicistronic expression so that the biomarker expression is not altered by the expression of the reporter.

It is then possible to efficiently and easily monitor the biomarker expression without interfering with its functional network. This approach provides more reliable and valuable information under normal physiological conditions, but more interestingly, under pathological conditions, under special diets, under stress conditions, etc.

Such models have been developed for the assessment of compound polarizing the immune response, detection of protective immune response, detection of effector functions (cytotoxic, antitumor, helper cells, repressor cells).

 

Case study: Monitoring of IL4 expression.


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Adapted from Mohrs et al. Immunity 2001. Analysis of type 2 immunity in vivo with a bicistronic IL4 reporter.

IL4 induces differentiation of naive Th cells to Th2 cells polarising the immune response toward a humoral effector response. IL4 remains the canonical marker of Th2 cells.

Reporter gene and IL-4 expressionExpression of the reporter gene does not interfere with IL4 expression.

Monitoring of IL4 gene expression is done by 3'UTR insertion of the reporter gene (bicistronic expression via IRES technology). Reporter expression is proportional to IL4 gene expression.
 

IL-4 expression monitoring

Reliable and Sensitive Monitoring of IL4 Expression

The reporter is only detected in IL4 expressing cells, enabling a reliable detection and monitoring of the IL4 expression.

GFP expression correlates with IL4 expression and allows monitoring of early events of an effector immunity type 2. Established by FACS analysis on activated splenocytes.
Although all culture conditions induce cell division (activation), GFP+ cells are detected only in Th2 polarizing culture conditions. In addition, GFP can be detected as early as 36h after the onset of the culture as well as prior to cell division (40-45h).
 

GFP expression to monitor IL-4 biological and effector function

Monitoring with Unmodified IL4 Activity

IL4 biological and effector function is preserved in GFP reporter mice. GFP expression enables a reliable monitoring of IL4 responses.

FACS analysis of freshly isolated cells from different organs.

Protection against Nocardia brasiliensis is Th2 mediated. An efficient Th2 priming results in worm expulsion within 10 days. Priming of CD4 cells was analyzed in lung, mesenteric lymph node (MLN), peripheric lymph node (PLN) and splenocytes. Apparition of IL4 producing cells is consistent with the worm trafficking and demonstrates that the presence of GFP does not alter the protective Th2 immunity.