Efficient ablation by immunotoxin-mediated cell targeting of the cell types that express human interleukin-2 receptor depending on the internal ribosome entry site

✍ Scribed by Tomoko Kobayashi; Yukiko Kida; Takeshi Kaneko; Ira Pastan; Kazuto Kobayashi

• Publisher: John Wiley and Sons
• Year: 2001
• Tongue: English
• Weight: 164 KB
• Volume: 3
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.208

✦ Synopsis

Background:

Immunotoxin-mediated cell targeting (imct) is a technique for conditional genetic ablation of specific cell types. imct provides a useful approach for generating animal models for human neurodegenerative disorders. the strategy of imct depends on the cytotoxic activity of antitac-based recombinant immunotoxins that selectively target cells expressing the human interleukin-2 receptor alpha-subunit (il-2ralpha). transgenic mice were generated that express the il-2ralpha under the control of an appropriate tissue-specific gene promoter, and they were treated with the recombinant immunotoxins resulting in the ablation of the target cell types. to restrict the expression of il-2ralpha transgene in the cell types of interest, it is useful to knock-in the il-2ralpha expression cassette into the specific marker gene locus with gene targeting. moreover, the knock-in of the il-2ralpha cassette located downstream of an internal ribosome entry site (ires) into the 3'-untranslated region of the marker gene enables il-2ralpha expression in the restricted cell types while preserving the intact marker gene expression. however, there is a possibility that ires-dependent expression of the receptor may be less efficient than cap-dependent expression.

Methods and results:

The efficiency of ires-dependent il-2ralpha expression and immunotoxin responsiveness of the cells expressing the receptor were examined. the il-2ralpha gene fused to green fluorescence protein (gfp) (il-2r/ gfp) was used as the target receptor. embryonic stem cell clones were isolated that carry two types of bicistronic vectors in which the il-2r/gfp fusion gene or the chloramphenicol acetyltransferase gene was connected upstream or downstream of ires. the expression level of il-2r/gfp protein in the cell clones was evaluated by gfp fluorescence detection and western blot analysis. the ires-dependent expression produced the same level of receptor protein as cap-dependent expression. the immunotoxin responsiveness of the cloned cells was evaluated by measuring the colony-forming efficiency in medium containing various amounts of a recombinant immunotoxin. the colony-forming efficiency of the cells expressing il-2r/ gfp through ires-dependent expression was reduced together with increasing immunotoxin concentration in a similar dose-dependent manner to the cells expressing the receptor through cap-dependent expression.

Conclusions:

The present results indicate that it is possible to effectively use the ires-dependent expression system for imct. the system permits expression of the target receptor in selective cell types by introducing the ires-driven expression cassette into the 3'-untranslated region of the marker gene locus.