Effective RNAi-mediated β2-microglobulin loss of function by transgenesis in Xenopus laevis.

To 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.