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.
PubMed ID: 23519478
PMC ID: PMC3603415
Article link: Biol Open.
Grant support: R24 AI059830 NIAID NIH HHS
Genes referenced: b2m gapdh myh4 myh6
Article Images: [+] show captions
|Fig. 1. In vivo b2m silencing in transgenic F0 Xenopus.(A) A map of the I-SceI- anti-b2msiRNA-GFP construct used to generate transgenic LG clones. (B) LG eggs were injected (Experimental) with 1×10−3 I-SceI units and 80 pg of I-SceI-b2msiRNA-GFP in a total volume of 10 nl per egg. RNA extracted from 7-day-old (stage 48) tadpoles was used to synthesize cDNA. Shown is RT-PCR analysis of experimental and control non-transgenic dejellied animals from the same batch (Dejellied controls) using primers specific for Xenopus b2m, class Ia, GAPDH and GFP. % KD is the percent silencing of b2m (β2-m) in transgenic larvae compared to the average expression in the control animals. −RT controls for all samples were negative. One representative experiment out of two is shown.|
|Fig. 2. Effective b2m silencing in vivo in transgenic F1 OB and LG Xenopus clones.RT-PCR was performed on RNA from the skin of young adults (2 months of age) OB F1 transgenic (GFP+) carrying either the anti-b2m shRNA (OB-Tg, 9 individuals), the scram shRNA transgene (Sc, 6 individuals) and RNA from skin of LG-6 anti-b2m shRNA transgenic (LG6-Tg, 9 individuals). RT-PCR analysis was performed using primers specific for Xenopus GAPDH and b2m. −RT controls for all samples were negative. The percent of b2m KD was determined by densitometry after normalization to GAPDH by comparing b2m expression of each transgenic to the average expression in the respective WT controls. Average ± s.d. of b2m is also indicated by filled symbols for each group of transgenic. Statistical significance between controls and transgenic animals was determined with a t-test.|
|Fig. 3. Xenopus LG and OB F1 progeny have downregulated cell surface expression of MHC class Ia molecules.F1 transgenic progeny were reared to adulthood and screened for GFP fluorescence. Frogs were bled and the blood (including both red blood cells and lymphocytes) was analyzed by flow cytometry for surface MHC class Ia using the Xenopus-specific anti-class Ia mAb TB17. 50,000 events were collected, gated on live cells (includes both red blood cells and lymphocytes) and analyzed using the FlowJo software. (A) LG transgenic F1 frog carrying the β2-m shRNA (thick black histogram) and LG age matched control (gray tinted histogram) are shown. (B) OB F1 transgenic anti-b2m shRNA (thick black histogram) and a control F1 transgenic scram shRNA frog (gray tinted histogram) are shown. Secondary antibody controls are shown as dashed histograms. The median fluorescence intensities (MFI) of both the transgenic and control frog are indicated. One representative individual of each group is shown.|
|Fig. 4. LG-6 clones with silenced b2m expression have reduced frequency of circulating CD5/CD8 T cells in the spleen and the blood.Spleen and blood were harvested from two transgenic LG-6 froglets with silenced b2m expression as well as an age matched LG-6 naïve animal. Cells were stained for CD5 and CD8 and were analyzed by flow cytometry. 10,000 events were collected and were gated on live leukocytes. Analysis was performed using the FlowJo software. Shown are dot plots of CD5 and CD8 expression with % of each population labeled.|
|Fig. 5. Effects of b2m knockdown on allograft rejection.OB and LG-6 anti-b2m shRNA transgenic and aged-matched controls adults were grafted with a MHC disparate J skin. At 8 days post-transplantation, pigment destruction is easily notable in control animals (*), whereas little rejection is visible in both transgenic recipients.|
|Fig. 6. In vivo b2m silencing results in delayed MHC disparate skin graft rejection.(A) Adult anti-b2m shRNA transgenic LG-6 clones (LG-6 Tg) and control naïve LG-6 animals (LG-6 C) grafted with a MHC disparate J skin. Skin graft rejection was monitored daily at 21°C until complete rejection. (B) Adult outbred frogs carrying either anti-b2m shRNA (OB β2-m shRNA) or scram shRNA (OB scram shRNA) transgenes were grafted with a MHC disparate J skin. Skin graft rejection was monitored at 21°C until complete rejection. Each group consisted of 3 animals and the graphs show the average skin graft rejection of the group. P values were determined using the Student's t-test.|
|Fig. 7. Decreased CD8 T cell infiltration in skin grafts from transgenic OB frogs with silenced β2-m.Transgenic outbred anti-b2m shRNA adults (B,D) or aged-matched control OB frogs (A,C) were grafted with a MHC disparate J skin. Eight days later grafts were harvested and were stained without fixation with a Xenopus specific anti-CD8 antibody (AM22). Scale bar: 20 µm.|
|Fig. 8. Transgenic OB tadpoles carrying anti-b2m shRNA are more susceptible to FV3 infections.(A) F0 transgenic tadpoles (stage 56, GFP+) carrying either a shRNA targeting b2m (Tg, 23 larvae) or WT (Control, 10 larvae) were infected with 5×106 PFU FV3 via the water route for 1 hour. (B) F0 transgenic tadpoles (stage 56, GFP+) carrying either a shRNA targeting b2m (Tg β2-m shRNA, 36 larvae) or a scrambled shRNA (Tg scram shRNA, 22 larvae) were infected with 5×106 PFU FV3 via the water route for 1 hour. Survival was monitored daily. P values were determined using the Student's t-test.|