Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

Abstract

The expression of the cellular form of the prion protein (PrP(c)) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrP(c) is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrP(c), a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrP(c) at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

Figure 1

Schematic representation of the bovine PrP gene and structure of the transgene used in this study. Exons (E) are numbered, and exon 1 and exon 2 are indicated by solid boxes. Within exon 3, the PrP ORF is shown by an open large box, and the mRNA 3′ UTR is represented by an open small box. The lengths of exons 1a, 1b, 2, and 3 are 53, 115, 98, and 4554 bp, respectively. The transcription initiation site is designated with a solid arrow (30). The positions of the two phages (λ1 and λ2) spanning the PrP gene are indicated. The primers used for the analysis of the 5′ UTR are shown by thin arrows (not to scale). The transgene encompasses 6.9 kb of the upstream sequences fused to the gfp reporter gene. GFP, gfp gene ORF; Poly(A), the simian virus 40 polyadenylylation signal. Positions are bp with respect to the transcription initiation site.

Figure 2

Coexpression of the two different bovine PrP 5′ UTR exons in 17 different tissues. RNA fractions were subjected to reverse transcription–PCR using primers specific to exon 1a and exon 3. The PCR products were analyzed by Southern blot hybridization with a ³²P-labeled oligonucleotide specific for exon 1a (A and B). The hybridization revealed two fragments (250 and 350 bp). Hybridization with a ³²P-labeled oligonucleotide specific for exons 3 or 2 gave identical results. Hybridization with an exon 1b-specific oligonucleotide revealed only the 350-bp fragment, as shown for brain and spleen (C). All tissues tested gave identical results (data not shown). Lu, lung; Br, brain; Ce, cerebellum; Ad, adrenal gland; Mu, skeletal muscle; He, heart; To, tonsil; Sp, spleen; Re, retina; On, optic nerve; Ln, lymph node; In, intestine; Sk, skin; Th, thymus; Me, mesentery; Li, liver; Ki, kidney. A and B differ by the autoradiography exposure conditions, 2 h at room temperature or at −80°C, respectively. Autoradiography in C was exposed overnight at −80°C.

Figure 3

Analysis of gfp and PrP expression from tg mice (L64) by fluorescence-activated cell sorter on peripheral blood leukocytes, gated for B (A) and T (B) lymphocytes. B cells were stained with R-phycoerythrin-conjugated anti-mouse CD19 antibody, T cells with Cy-chrome-conjugated anti-mouse CD3 molecular complex antibody (Becton Dickinson), and PrP-expressing cells with biotinylated anti-PrP antibody (8G8). The percentage of positive cells is indicated in the upper right corner.

Figure 4

Analysis of reporter gene expression in PrP-gfp tg mice (L64). (A) Coronal section of cerebellar cortex in which the transgene is expressed in Purkinje neurons. (×200.) (B) Section of the wall of the small intestine showing fluorescence in the ganglion cells in the enteric nervous system. (×200.) (C) Fluorescence picture of the foot from the gfp tg mice (Lower) and their littermate controls (Upper). (D and E) Sections of thymus showing fluorescence in the epithelial cells of the medulla (m) around the Hassal's corpuscles (arrow). c, cortex. [×40 (D) and ×200 (E).] (F) Thymus section stained with eosin and hematoxylin. E and F are serial sections. (G) Sections of renal cortex showing fluorescence in the endothelial cells of the capillaries around the tubules. (×200.) (H and I) Sections of small intestine of gfp-tg mice (H) and wild-type mice stained with anti-PrP 8G8 antibody (1:200) (I). (×200.)