Expression of a dominant negative IFN‐γreceptor on mouse oligodendrocytes

Abstract

The interferon-gamma (IFN-gamma) receptor is expressed by all nucleated cells, and binding of its cognate ligand, IFN-gamma, induces a wide variety of biological functions. Transgenic mice expressing a dominant negative IFN-gamma receptor 1 (IFN-gammaR1DeltaIC) on oligodendrocytes under control of the myelin proteolipid protein promoter are described. The mRNA encoding the transgene was only detected in the nervous system and protein expression was confirmed by immunohistochemistry. Transgenic receptor expression does not alter myelination and the mice exhibited no clinically apparent phenotype. Consistent with the restricted nervous system expression of the transgene, no alterations in peripheral immune responses were detected. Flow cytometric analysis demonstrated constitutive expression of both the IFN-gammaR1DeltaIC transgene and the endogenous IFN-gamma receptor 2 at high levels on oligodendrocytes derived from the transgenic mice. These oligodendrocytes also exhibited decreased STAT1 phosphorylation in response to IFN-gamma, confirming dominant negative transgene function. Transgenic mice in which oligodendrocytes have a diminished ability to respond to IFN-gamma showed delayed virus clearance from oligodendroglia compared with wild-type mice. This model will allow evaluation of oligodendrocyte responses to this critical cytokine during CNS inflammation.

Suppressor of Cytokine Signaling 1 Expression Protects Oligodendrocytes from the Deleterious Effects of Interferon-γ

“…1 A). The PLP expression cassette (a kind gift from Dr. Wendy Macklin, Cleveland Clinic, Cleveland, OH) has been described previously and has been used for oligodendrocyte-specific expression of a number of transgenes (Wight et al, 1993;Fuss et al, 2000;Doerflinger et al, 2003;Gonzales et al, 2005). We used an SOCS1 cDNA clone (kindly provided by Douglas Hilton, Walter and Eliza Hall Institute, Melbourne, Victoria, Australia) (Starr et al, 1998) that contained a Flagepitope sequence that served as a marker for SOCS1 expression in PCRbased or anti-Flag antibody-based detection methods (Einhauer and Jungbauer, 2001).…”

Expression of a Myelin Proteolipid Protein (Plp)-lacZ Transgene is Reduced in both the CNS and PNS of Plp jp Mice

“…The PLP(+)Z transgene contains the proximal 2.4 kb of 5′-flanking DNA, exon 1, intron 1 and the first 37 bp of exon 2 of the Plp gene, and encodes a fusion protein between the first 13 amino acids of PLP and β-galactosidase (β-gal), which is trafficked to the myelin membrane [12]. A series of transgenic animals have subsequently been derived that further corroborate the ability of these Plp sequences to promote transgene expression, and to serve as a reliable readout of endogenous Plp gene activity [13][14][15][16][17][18], with inclusion of Plp intron 1 sequences being important [19]. In the current studies, the PLP(+)Z transgene was crossed into the Plp jp background and used to monitor the effects of the Plp jp mutation on Plp promoter activity via lacZ reporter gene expression.…”

Vaccine-Induced Memory CD8+ T Cells Cannot Prevent Central Nervous System Virus Reactivation

“…This preempts a direct comparison of in vivo T cell function during the early stages of recrudescence. Furthermore, earlier down-regulation and reduced class I expression on oligodendrocytes, compared with microglia, before recrudescence in 2Ј mice may reflect reduced IFN-␥ mediated signaling (28). These findings highlight the concept that release of CD8 ϩ T cell effector molecules such as perforin and IFN-␥ is not only strictly dependent upon recognition of cognate Ag-MHC structures in vitro, but also in situ (39,42).…”

“…Briefly, brains were removed from mice perfused with PBS, homogenized in RPMI 1640 containing 25 mM HEPES (pH 7.2), using Tenbroek homogenizers and adjusted to 30% Percoll (Amersham Biosciences). To isolate oligodendroglia, brains and spinal cords were obtained from perfused mice and digested with 0.25% trypsin as previously described (28). CMC were concentrated by centrifugation at 800 ϫ g for 25 min at 4°C onto a 70% Percoll cushion.…”

“…CD45 (30-F11; BD Pharmingen) expression was used to distinguish bone marrow-derived infiltrating cells (CD45 high ), CNS resident microglia (CD45 low ), and other CNS resident cells (CD45 Ϫ ). Oligodendrocytes in the CD45 Ϫ population were identified using biotinylated O4 mAb followed by avidin/allophycocyanin (BD Pharmingen) as described (28). The gating strategy distinguishing infiltrating bone marrow-derived CD45 high leukocytes from CD45 low microglia and CD45 Ϫ O4 ϩ oligodendrocytes is shown in Fig.…”