Identify genes that participate in IFN-signaling pathway by screening ENU mutagenized mice
Date Issued
2006
Date
2006
Author(s)
Wei, Pei-Chi
DOI
en-US
Abstract
Interferons (IFNs) are cytokines that have immunmodulatory, anti-proliferation, and antivirus ability. Although IFNs activate JAK-STAT pathway, several studies suggest that there may be some alternative regulatory mechanisms of IFN signaling. To further study the fine regulatory mechanisms and to discover those undefined IFN signaling machineries, we have taken a genetic approach to screen ENU-mutagenized mice that displayed altered IFN responses.
We have developed a two-step screening strategy, first by flow cytometry to monitor surface markers like MHC molecules that are tightly regulated by IFNs and then by real-time QPCR for different sets of IFN-inducible genes to screen mice for abnormal responses. We have screened some 2800 mice from 134 pedigrees and find that three pedigrees shown impaired IFNs responses. They are 117i, which shows hypo-responsiveness to IFNα, 243g and 248k, which have hypo-responsiveness to both IFNγ and IFNα. Using RT-QPCR, we have shown that 117i pedigree fails to induce ISRE-containing genes such as OAS and PKR in response to IFNα. However, the response to IFNγ is normal. By crossing the mutant mice to wild type mice, we have demonstrated that the hypo-responsiveness to IFNα in 117i mice is inheritable and is a recessive trait because no altered IFNα response is found in the offspring of a cross between wild type and deviant mice. However, we do observe mice with hypo-responsiveness in the offspring from a cross between two F1 (117i x wild type) mice that do not display the phenotype.
The development of hematopoietic cells appears to be normal in the mutant mice as T cell, B cell, monocyte and granulocyte populations in bone marrow, thymus, spleen and PBL of mutant mice are comparable to that of wild type (WT) mice. In addition, the susceptibility of high dose LPS-induced toxic shock in the mutant mice is also similar to that of WT mice. In searching for possible mechanisms accounting for the impaired response to IFNα, we perform Western blotting using antibodies to STAT1, STAT2, and STAT3. Interestingly, we found that STAT2 protein is not detected in either PBL or splenocytes of the mutant mice, suggesting that the loss of STAT2 in mutant mice may result in the altered response to IFNα but not IFNγ. However, mRNA of STAT2 is still present and inducible, though at lower level, in mutant cells in response to IFNα, suggesting that the absence of STAT2 may lie in a post-transcriptional or translational mechanism. Genetic mapping and sequencing of STAT2 genome should be able to allow us to pinpoint the gene that causes the altered IFN response.
Subjects
干擾素
IFN
ENU
novel
Type
other
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