Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids

Abstract

Extracellular vesicles (EVs) play an important role in the transfer of biomolecules between cells. To elucidate the intercellular transfer fate of EVs in vivo, we generated a new transgenic (Tg) rat model using green fluorescent protein (GFP)-tagged human CD63. CD63 protein is highly enriched on EV membranes via trafficking into late endosomes and is often used as an EV marker. The new Tg rat line in which human CD63-GFP is under control of the CAG promoter exhibited high expression of GFP in various body tissues. Exogenous human CD63-GFP was detected on EVs isolated from three body fluids of the Tg rats: blood serum, breast milk and amniotic fluid. In vitro culture allowed transfer of serum-derived CD63-GFP EVs into recipient rat embryonic fibroblasts, where the EVs localized in endocytic organelles. These results suggested that this Tg rat model should provide significant information for understanding the intercellular transfer and/or mother-child transfer of EVs in vivo.

Figure 1. Generation of CAG/human CD63-GFP transgenic (Tg) rats.

(a) Structure of the transgene construction. The transgene was constructed using human CD63-copGFP under control of the CAG promoter. (b) Image of rat embryonic stem cells (rESCs) transfected with the CAG/human CD63-GFP gene. The cultured rESCs expressed GFP. (c) Blastocysts after microinjection of the transfected rESCs. The arrow indicates rESC adherence to the inner cell mass (ICM). BF: bright field. Scale bars = 100 μm. (d) Adult female chimaeric rat from Wister-derived rESC (white-coated) injection into LEA blastocysts (brown-coated). White patches were present in the face (arrowhead). Two Tg offspring (white-coated) from mating a female chimaeric rat with a Wistar wild type (Wt) male (arrows). (e) Genotyping by PCR analysis of the extracted DNA from ear snips of the offspring. B: brown coat colour, W: white coat colour, V: CAG/human CD63-GFP vector, and M: size marker.

Figure 2. Human CD63-GFP expression analysis in Tg rats (Wister-esTgN(CAG/CD63-GFP)3NCCRI).

(a) Pictures of main organs from Tg offspring (i–xiii: bright field, i’–xiii’: GFP, and xiii”: merged). GFP-negative (i and i’) and GFP-positive (ii and ii’) offspring were littermate. The heart, kidneys and stomach showed especially high fluorescent signals (xiii”). (b) Western blotting for endogenous rat CD63 and exogenous human CD63 in tissue lysates from GFP-negative (GFP−) and GFP-positive (GFP+) offspring. Ctx: cortex, cbl: cerebellum, and hip: hippocampus.

Figure 3. Human CD63-GFP localization and extracellular vesicle (EV) analysis in the primary fibroblast cells obtained from the caudal vertebrae of Tg rats.

(a) Localization of human CD63-GFP in the cultured Tg rat cells. Immunostaining indicated the co-localization of GFP with human CD63 (upper panels) and with rat CD63-positive signals (lower panels) around nuclei (blue). Scale bars = 50 μm. (b) Size distribution of the EVs isolated from the conditioned medium of Wt and Tg rat cells was determined using a NanoSight system. (c,d) The relationship between ceramide and the secretion of EVs. The intracellular rat CD63-positive signals and GFP signals were increased after treatment with 10 μM GW4869, a neutral sphingomyelinase (nSMase) inhibitor, for 24 hours (c). Western blotting showed a GW4869-dependent decrease of EV markers (rat CD63 and flotillin-1) and human CD63-GFP in the isolated EVs (d; left). However, the expression levels of rat CD63 and human CD63-GFP in the cell lysates were not changed by GW4869 (d; right). β-actin was used as a loading control. Both generation and protein composition of the EVs did not show an apparent change by CD63-GFP overexpression (Supplementary Fig. 5).

Figure 4. Characterization of the EVs isolated from three body fluids of Wt and Tg rats.

(a–c) Size distribution of EVs from serum (a), breast milk (b) and amniotic fluid (AF) (c) determined using a NanoSight system. (d) Electron microscopy (EM) images of EVs. Scale bars = 200 nm. AF samples were collected from pregnant Tg rats at embryonic day 16–17 (E16–17) after mating with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses.

Figure 5. Analysis of the EVs labelled with human CD63-GFP in the three body fluids.

(a,b) Western blotting analysis of the EVs isolated from serum (a), breast milk and AF (b) of Wt and Tg rats for flotillin-1, rat CD63, human CD63 and copGFP. AF samples were collected from pregnant Tg rats at E16–17 after mating with Wt males. GFP−: GFP-negative foetuses. GFP+: GFP-positive foetuses. (c) Immunoelectron microscopy images of serum-derived EVs from Wt and Tg rats using anti-human CD63 antibody (10 nm gold particles). Scale bars = 200 nm.

Figure 6. Uptake of serum-derived EVs by rat embryonic fibroblast cells (REFs).

(a) EVs of Wt rats, Tg rats and vehicle PBS (−) as a control were prelabelled with PKH67. Then, REFs were incubated with PKH67-labelled EVs for 11 hours. The incorporated EVs in the recipient REFs were detected by PKH67 (green) and anti-human CD63 (red). The Tg EVs are also shown as magnified views (white square region) with different colours (PKH67: blue, human CD63: red, nuclei: gray). Scale bars = 20 μm. (b) Images of co-localization of EVs with LAMP1 in REFs. EVs of Wt (with PKH67) and Tg rats were incubated with REFs for 10 hours, and the EVs from Tg rats were detected using an antibody to human CD63. Magnified views (arrows) are shown in the insets. Scale bars = 25 μm. Nuclei were counterstained with Hoechst 33342 (blue).