Effective RNAi-mediated β2-microglobulin loss of function by transgenesis inXenopus laevis

Abstract

SummaryTo impair MHC class I (class I) function in vivo in the amphibian Xenopus, we developed an effective reverse genetic loss of function approach by combining I-SceI meganuclease-mediated transgenesis with RNAi technology. We generated transgenic outbred X. laevis and isogenetic laevis/gilli cloned lines with stably silenced expression of β2-microglobulin (b2m) critical for class I function. Transgenic F1 frogs exhibited decreased surface class I expression on erythrocytes and lymphocytes, decreased frequency of peripheral CD8 T cells and impaired CD8 T cell-mediated skin allograft rejection. Additionally, b2m knockdown increased susceptibility to viral infection of F0 transgenic larvae. This loss of function strategy offers new avenues for studying ontogeny of immunity and other developmental processes in Xenopus.

Nonclassical MHC class I-dependent invariant T cells are evolutionarily conserved and prominent from early development in amphibians

“…Given the XNC10 gene expression by larval thymocytes at the onset of thymic organogenesis and the presence of splenic XNC10-T + -reactive cells during early development, we hypothesized that XNC10 is required for iVα6T cell differentiation. To test this hypothesis, we used our recently developed reverse-genetic loss-of-function approach, which combines RNAi and I-SceI meganuclease-mediated transgenesis (17). A key advantage of this technique is that the high efficiency and nonmosaic transgene expression permit the direct use of F0 animals (17).…”

“…To test this hypothesis, we used our recently developed reverse-genetic loss-of-function approach, which combines RNAi and I-SceI meganuclease-mediated transgenesis (17). A key advantage of this technique is that the high efficiency and nonmosaic transgene expression permit the direct use of F0 animals (17). Outbred F0 transgenic tadpoles (stage 53, 25 d postfertilization) were screened for uniform nonmosaic GFP expression, and XNC10 knockdown was assessed by quantitative PCR (qPCR) compared with age-matched controls (Fig.…”

“…Although Xenopus tadpoles are not as resistant to FV3 infection as adults, they do mount antiviral immune responses (26). We previously reported that beta 2 microglobulin (b2m) silencing, which impairs both class Ia and XNCs surface expression, increased susceptibility to FV3 infection in young tadpoles (17). We interpreted these data as evidence for a critical role of class Ib in antiviral immunity during early ontogeny, when class Ia expression is suboptimal (17).…”

“…We previously reported that beta 2 microglobulin (b2m) silencing, which impairs both class Ia and XNCs surface expression, increased susceptibility to FV3 infection in young tadpoles (17). We interpreted these data as evidence for a critical role of class Ib in antiviral immunity during early ontogeny, when class Ia expression is suboptimal (17). The marked increase in susceptibility to FV3 infection resulting in a rapid increase in mortality within a few days postinfection obtained by specifically silencing one single class Ib gene, XNC10, shows that class Ib molecules play a role in antiviral immunity in larvae.…”

“…We hypothesized that XNC10 is critically involved in Xenopus iT cell biology. Accordingly, we generated an X. laevis class Ib tetramer and developed an effective reverse-genetic loss-offunction approach by combining I-SecI meganuclease-mediated transgenesis with RNAi technology (17).…”