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


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.
Kobayashi
T
T
Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Japan.
Kida
Y
Y
Kaneko
T
T
Pastan
I
I
Kobayashi
K
K
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D018390:Gene Targeting; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D000922:Immunotoxins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D012315:RNA Caps / Q000235:genetics; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics*; D013234:Stem Cells / Q000166:cytology
D000922:Immunotoxins; D008164:Luminescent Proteins; D012315:RNA Caps; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015500:Chloramphenicol O-Acetyltransferase