Exomap1 mouse: a transgenic model for in vivo studies of exosome biology

Abstract

Exosomes are small extracellular vesicles (sEVs) of ~30-150 nm in diameter that have the same topology as the cell, are enriched in selected exosome cargo proteins, and play important roles in health and disease. To address large unanswered questions regarding exosome biology in vivo, we created the exomap1 transgenic mouse model. In response to Cre recombinase, exomap1 mice express HsCD81mNG, a fusion protein between human CD81, the most highly enriched exosome protein yet described, and the bright green fluorescent protein mNeonGreen. As expected, cell type-specific expression of Cre induced the cell type-specific expression of HsCD81mNG in diverse cell types, correctly localized HsCD81mNG to the plasma membrane, and selectively loaded HsCD81mNG into secreted vesicles that have the size (~80 nm), topology (outside out), and content (presence of mouse exosome markers) of exosomes. Furthermore, mouse cells expressing HsCD81mNG released HsCD81mNG-marked exosomes into blood and other biofluids. Using high-resolution, single-exosome analysis by quantitative single molecule localization microscopy, we show here that that hepatocytes contribute ~15% of the blood exosome population whereas neurons contribute <1% of blood exosomes. These estimates of cell type-specific contributions to blood EV population are consistent with the porosity of liver sinusoidal endothelial cells to particles of ~50-300 nm in diameter, as well as with the impermeability of blood-brain and blood-neuron barriers to particles >5 nm in size. Taken together, these results establish the exomap1 mouse as a useful tool for in vivo studies of exosome biology, and for mapping cell type-specific contributions to biofluid exosome populations. In addition, our data confirm that CD81 is a highly-specific marker for exosomes and is not enriched in the larger microvesicle class of EVs.

Keywords: CD81; Cre recombinase; EV; H11; exosome biogenesis; extracellular vesicle; mNeonGreen; tdTomato.

Figure 1.. Design, genetics, and red fluorescence of exomap1^+/− mice.

(A) Line diagram of the exomap1 transfer vector. (B, C) Fluorescence micrographs of DAPI-stained HEK293 cells transfected with (B) pFF077 or (C) pFF077 + pJM776 that had been fixed and stained with DAPI. Bar, 10 um. (D) Anti-CD81 immunoblot of cell and exosome fractions of (left lanes) HEK293 cells and (right lanes) HEK293 cells expressing HsCD81mNG. MW size standards are in kDa. (E) Ethidium bromide-stained agarose gel electropherogram of genomic DNA (gDNA) PCR products generated using gDNAs extracted from (control) non-transgenic mouse and the (Fo) exomap1 founder mouse using the H11 locus 5’ primer and the CAG promoter 3’ primer, showing the 447 bp product that is diagnostic for the exomap1 transgene. MW size markers are in bp. (F) Ethidium bromide-stained agarose gel electropherograms of PCR reaction carried out with gDNAs extracted from the tails of six F1 progeny from a cross between the (Fo) founder mouse and a non-transgenic control mouse, as well as gDNAs from the founder mouse and a non-transgenic control mouse. Upper panel shows products obtained using the H11 locus 5’ primer and the CAG promoter 3’ primer. Lower panel shows products obtained using the bGH-pA 5’ primer and the FRTR2 3’ primer, with an expected exomap1 transgene-specific product of 311 bp. MW size markers are in bp. (G Fluorescence micrograph of three F1 mice, illuminated with green light and imaged with a red filtered camera. White arrows point to heads to four exomap1^+/ carrier mice, readily identifiable by their red fluorescent ears, feet, and tails. Black bordered arrows point to the heads of three non-transgenic littermates. Bar, 1 cm.

Figure 2.. Cre-triggered expression of HsCD81mNG in exomap1 mice.

(A) Fluorescence micrograph showing red and green fluorescence of an oocyte and a cluster of attached cumulus cells from an exomap1::Zp3-Cre mouse. Bar, 100 um (B) Fluorescence micrograph of oocytes and granulosa cells from exomap1::Cyp19a1-Cre mice showing lack of exomap1 transgene expression in granulosa cells and strong exomap1 (MTS-tdTomato) expression in oocytes. White arrowheads point to oocytes. Bar, 100 um. (C) Fluorescence micrograph of DAPI-stained section through an exomap1::Cyp19a1-Cre mouse ovary, showing the absence of exomap1 transgene expression in granulosa cells of developing follicles (blue DAPI positive only) as well as strong exomap1 transgene expression in corpus luteum cells, with most cells expressing HsCD81mNG while some expressed MTS-tdTomato and no HsCD81mNG. Bar, 100 um. (D-I) Flow cytometry histograms of CD45+, CD11b+ cells collected from the blood of (D, E) control mice, (F, G) exomap1^+/ mice, and (H, I) exomap1::LysM-Cre animals showing plots of cell number vs brightness of (D, F, H) HsCD81mNG fluorescence and (E, G, I) MTS-tdTomato fluorescence. (J-K) Fluorescence micrographs of a cross section through the skeletal muscle of a tamoxifen-induced exomap1::HSA-MCM mouse stained with DAPI, sectioned, showing the images for (J) HsCD81mNG fluorescence and DAPI, (K) MTS-tdTomato fluorescence and DAPI. (L HsCD81mNG, MTS-tdTomato, and DAPI. Bar, 100 um.

Figure 3.. Hepatocytes load HsCD81mNG into mouse exosomes.

(A, B) Fluorescence micrographs of liver sections from (A) a non-transgenic control mouse and (B) an exomap1^+/− mouse, stained with DAPI and imaged for (blue) DAPI and for (red) MTS-tdTomato fluorescence. Bar, 100 μm in A and 20 μm in B. (C) Fluorescence micrograph of a liver section obtained from an exomap1^+/ mouse infected i.v. with AAV8/TBG-Cre virus, stained with DAPI, and imaged for (blue) DAPI and (green) HsCD81mNG fluorescence. Bar, 100 μm. (D) Fluorescence micrograph of a liver section obtained from an exomap1^+/ mouse infected i.v. with AAV8/TBG-Cre virus, stained with DAPI, and imaged for (blue) DAPI, (green) HsCD81mNG fluorescence, and (red) MTS-tdTomato fluorescence. Bar, 20 μm. (E) Fluorescence micrograph of a brain section obtained from an exomap1^+/ mouse infected i.v. with AAV8/TBG-Cre virus, stained with DAPI, and imaged for (blue) DAPI, (green) HsCD81mNG fluorescence. Bar, 20 nm. (F-I) Histograms of qSMLM data that we collected for raw plasma exosomes immunopurified on coverslips derivatized with antibodies specific for (F, G) MmCD81 or (H, I) HsCD81, from (F, H) an exomap1+/− mouse infected i.v. with AAV8/TBG-Cre virus and (G, I) an exomap1+/− mouse, that had been stained witheither (red) AF647-labeled anti-mouse tetraspanins (MmCD9, CD63, and CD81) or (blue) AF647-labeled anti-HsCD81. TSPAN/EV_Av denotes the average number of detected target markers per vesicle, CV denotes the coefficient of variation, and D_Av denotes the mean diameter. The box and whisker plot below histograms denote the mean (x), median (line), interquartile range (box), and point beyond 1.5-times the interquartile range (hollow dots). P values are included elsewhere (Table S1). (J-M) Fluorescence image overlays of (red dots) qSMLM localizations of bound surface tetraspanins and (greyscale) intrinsic HsCD81mNG fluorescence of raw plasma exosomes collected from an AAV8/TBG-Cre-infected exomap1^+/− mouse that had been affinity purified on (J, M) anti-MmCD81 or (K, L) anti-HsCD81 antibodies and stained with (J, K) AF647labeled anti-HsCD81 antibody or (L, M) AF647-labeled antibodies to mouse exosomal tetraspanins. Bar, 500 nm.

Figure 4.. Activation of the exomap1 transgene in brain and neurons.

(A-C) Blue/green fluorescence micrographs of DAPI-stained brain sections of exomap1^+/− mice injected with AAV5/Rpe65-Cre virus, showing HsCD81mNG fluorescence in cells of (A) the corpus callosum and hippocampus, (B) posterior commissure, and (C) lateral ventricle. Bar in A, 100 um; Bar in B and C, 50 um. (D, E) Fluorescence micrographs of DAPI-stained brain sections of exomap1::Dat-Cre mice showing HsCD81mNG fluorescence in (D) cells of the ventral tegmental area and (E) the axons in the striatum. Bar in A, 50 um; bar in B, 25 um. Cartoons depicting the brain location of these sections are presented to the right of each micrograph, and were generated in BioRender. (F) NTA histograms of raw CSF samples that had been passed through a 200 nm pore diameter size filter, showing (left histograpm) the size distribution profile of all CSF sEVs and (right histogram) the size distribution profile of mNeonGreen-positive sEVs. (G) Anti-HsCD81 immunoblot of brain protein extracts from control mice, exomap1^+/− mice, exomap1::Camk2a-Cre mice. MW size standards are in kDa. (H) Anti-HsCD81 immunoblot of kidney, liver, and brain protein extracts exomap1::Camk2a-Cre mice probed with antibodies specific for HsCD81. MW size standards are in kDa. (I-N) Micrographs showing anti-HsCD81immunohistochemical staining in tissue sections of (I, L) kidney, (J, M) liver, and (K, N) brain from (I-K) exomap1::Camk2a-Cre mice and (L-N) exomap1^+/ mice. Bar, 100um. (O, P) Histograms of qSMLM data for raw plasma samples collected from (O) exomap1::Camk2a-Cre mouse or (P) exomap1^+/ mouse, immunopurified on coverslips functionalized with (red) anti-MmCD81 antibody or (blue) anti-HsCD81 antibody, and stained with a cocktail of AF647-labeled antibodies to exosomal tetraspanins. TSPAN/EV_Av denotes the average number of detected target markers per vesicle, CV denotes the coefficient of variation, and D_Av denotes the mean diameter. The box and whisker plot below histograms denote the mean (x), median (line), interquartile range (box), and point beyond 1.5-times the interquartile range (hollowdots). (Q, R) Histograms of qSMLM data collected for raw CSF samples collected from (Q) exomap1::Camk2a-Cre mouse or (R) exomap1+/− mouse, immunopurified on coverslips functionalized with (red) anti-MmCD81 antibody or (blue) anti-HsCD81 antibody, and stained with a cocktail of AF647-labeled antibodies to exosomal tetraspanins. TSPAN/EV_Av denotes the average number of detected target markers per vesicle, CV denotes the coefficient of variation, and D_Av denotes the mean diameter. The box and whisker plot below histograms denote the mean (x), median (line), interquartile range (box), and point beyond 1.5-times the interquartile range (hollow dots). P values are included elsewhere (Table S1).