A novel vector for transgenesis in the rat CNS

VISTA software (http://​genome.​lbl.​gov/​vista/​index.​shtml) was used to align the rat Prnp locus with defined intronic and exonic regions (~23 kb; University of California, Santa Cruz, Genome Browser assembly ID: rn6; Entrez Gene: 24686) with mouse and SHa Prnp. Conserved regions were defined using a scanning 100 bp window with an identity greater than 50%. A rat bacterial artificial chromosome (BAC) template (Children’s Hospital Oakland Research Institute, CH230-380 M13) containing the rat Prnp locus was used for polymerase chain reactions (PCR) to amplify two fragments. The first fragment is Region I, which contains 6 kb of upstream sequence, to include elements of the rat Prnp promoter, Exon 1, Intron 1, and Exon 2. The second fragment, based on Region III, was PCR amplified; the open reading frame (ORF) of E3 was removed and a unique XhoI site was introduced in its place followed by the 3’UTR and 2.3 kb of downstream sequence. Importantly, we incorporated 15 bp In-Fusion flanking homology arms in Regions I and III and a linearized pUC19 backbone. With the addition of In-Fusion enzyme (Clonetech), all three fragments were combined to generate the RaPrnp vector. PE300 sequencing of the cos.Tet vector was done on a MiSeq instrument (Illumina) by the Mouse Biology Program at the University of California, Davis.

The RaPrnp vector was digested with XhoI and gel purified. Next, LacZ-T2A or T2A-EGFP fragments were PCR amplified from pGfaABC1D-nLac [9] and p799-IRES-EGFP (a gift from Jonathan Rubenstein) vectors, respectively. A dual LacZ-T2A-EGFP reporter cassette was created by overlap PCR. The rat Prnp ORF was PCR amplified from the original rat BAC template. Importantly, we generated flanking homology arms for the LacZ-T2A-EGFP and rat Prnp ORF to the XhoI linearized RaPrnp vector. Finally, In-Fusion cloning was used to introduce the LacZ-T2A-EGFP (LacZ/EGFP) cassette or rat Prnp ORF into the RaPrnp vector. Primer sequences used to amplify products are listed in Online Resource, Additional file 1: Table S1.

One μg of RaPrnp-LacZ/EGFP plasmid was transfected via X-tremeGENE HP DNA transfection reagent (Roche) into ~3 × 10⁵ cells per well in a six-well dish. Two days post-transfection, live EGFP fluorescence was captured by an EVOS Cell Imaging System using a 20× objective.

Methods supporting the preparation and generation of Tg rats were modified from Filipiak et al. [4]. Sprague Dawley (SD) rats were purchased from Charles River and allowed to acclimatize for 3–5 days in our facility prior to hormonal treatment. Rats were kept on a 12-h light/dark cycle (lights on 2 a.m. to 2 p.m.). Recipient SD female rats (9–10 weeks old) were synchronized for estrus  by injection with 0.2 ml (40 μg) Luteinizing Hormone-Releasing Hormone Analog (SIGMA, L4513) at 8 a.m. 4 days before mating with vasectomized SD males (2–8 months old) to induce pseudopregnancy. Pseudopregnancy was confirmed the following day by detecting the remains of a mating plug in recipients by an otoscope. Donor SD females (26–28 days old) were superovulated by injecting 0.2 ml (20 IU) of pregnant mare serum gonadotropin at 8 a.m. 2 days before mating followed by 0.2 ml (30 IU) human chorionic gonadotropin at 10 a.m. immediately before mating with donor males (2–8 months old). The day after mating, donor females were examined for the presence of mating plugs and euthanized by CO₂ followed by cervical dislocation. Oviducts were collected and placed in room temperature M2 media (Sigma Aldrich, M7167); the cumulus mass was released by tearing the ampulla. To detach the cumulus cells from the embryos, the cumulus mass was transferred to a drop of M2 containing hyaluronidase (1 mg/ml, Sigma, H4272) for 5–7 min. Embryos were serially washed in 5–7 drops of M2 media and then transferred into pre-equilibrated M16 media (Sigma Aldrich, M7292) and incubated at 37 °C; 5% CO₂ for 60 min. RaPrnp-LacZ/EGFP or RaPrnp-PrP transgenes were excised with NotI and gel purified to remove pUC19 backbone sequences. Gel-extracted fragments were purified with a Zymoclean large fragment DNA recovery kit (Zymo Research, D4046) and eluted into EmbryoMax injection buffer (Milipore, MR-095-10F). DNA was dialyzed on a DNA dialysis filter membrane (Millipore, VSWP02500) for 60–90 min in water for embryo transfer (Sigma, #W1503). A Nanodrop spectrophotometer (Thermo Scientific) was used to assess the concentration and purity (260/280 ratio of ≥1.8 and 260/230 ratio of 1.8–2.2) of dialyzed DNA sample. A sample of the resulting DNA was run on a 0.8% agarose gel to confirm a single band was visible. Transgenes were microinjected at 1–2 ng/μl into the pronuclei of one-cell stage rat zygotes. Because the cellular and pronuclear membranes of rat zygotes are highly elastic, microinjection needles were pulled longer and thinner (0.4–0.6 μm tip with 10 mm taper) than needles used for microinjection into mouse zygotes. Following microinjection, viable rat embryos were incubated in pre-equilibrated M16 media at 37 °C; 5% CO₂ for 30–60 min and transferred into the oviducts of pseudopregnant recipient females. All recipient females were administered analgesics, including meloxicam 2 mg/kg subcutaneously, sustained-release (SR) buprenorphine 1 mg/kg, and a local block of bupivacaine ≤ 7 mg/kg perioperative. The meloxicam and SR buprenorphine were continued pro re nata. Pups were screened for the presence of RaPrnp-LacZ/EGFP or RaPrnp-PrP transgenes by PCR with a RaPrnp forward genotyping primer and either a LacZ-T2A-EGFP or rat Prnp ORF reverse genotyping primer (Online Resource, Additional file 1: Table S1). Potential founder animals were mated to WT SD rats to establish the lines.

Rat genomic DNA (gDNA) was purified from ear or tail samples from Tg(RaPrnp-PrP) rats and diluted to 100 ng/μl. One μg of gDNA was digested with MseI (New England Biolabs R0525L) for 1 h at 37 °C. PCR reactions were set up with digested gDNA, ddPCR SuperMix: non-UTP (BioRad #1863024), Prnp-FAM (BioRad #10042958) target, and Rpp30-Hex (BioRad #10042961) reference copy number assay kits. PCR reactions and droplet reader oil (BioRad #1863004) were combined and then added to a QX200 droplet generator instrument (BioRad). Droplets underwent thermocycling in a C1000 instrument (BioRad) and read via a QX200 droplet reader. Copy number variation (CNV) analysis was done using QuantaSoft software (BioRad) by comparing the concentration of Prnp target (A) to the concentration of the Rpp30 (B) reference loci in gDNA samples. N_B = refers to 2 copies of Rpp30 in the rat genome. CNV = A/B*N_B. Founder transgene copy number was determined by the subtraction of WT Prnp copies (2) from the total Prnp copy number derived from the ddPCR assay.

All rats were examined at least once per day by a trained animal health technician for general appearance, activity level, hydration, body condition, abnormal posture, porphyrin staining, nature of ambulation, respiration quality, and qualitative food and water intake. A licensed veterinary technician or a veterinarian examined animals displaying any abnormal clinical sign. These included ataxia, bradykinesia, lethargy, hair loss, moribund state, porphyrin staining, clasping, and poor hair coat. Clinically sick animals were gently prodded to assess quality of ambulation and were briefly suspended by the tail to perform the hind-leg clasping reflex. Animals displaying two or more clinical signs were identified, and if their clinical condition did not change or deteriorated within 24 h, then the animal(s) was euthanized to define the incubation period in these studies.

Formalin-fixed hemi-brains were coronally coarse-cut and embedded into a paraffin block. After paraffin processing and embedding, sections were cut using a microtome set at an 8 μm thickness. Brain sections were then mounted on positively charged glass slides. These were then deparaffinized and stained with hematoxylin and eosin (H&E) or processed for immunohistochemistry. For immunofluorescent procedures, after removal of paraffin with xylenes and a graded series of alcohols, tissue sections were subjected to antigen retrieval with hydrolytic autoclaving (121 °C for 10 min in citrate buffer). After antigen retrieval, sections were incubated in 10% normal goat serum (Vector Laboratories, Inc., Burlingame, CA) made in 0.1 M PBS containing 0.2% Tween (PBST) for 1 h. After washing in PBST, slides were incubated in a primary antibody cocktail containing a 1:250 dilution of a rabbit PrP antibody (Abcam, ab52604) and 1:500 dilution of chicken GFAP (Abcam, ab4674) overnight at room temperature. The following day, sections were washed in PBST and incubated in a cocktail of specific secondary antibodies including goat anti-rabbit Alexa Fluor 488 (Thermo Fisher, Waltham, MA) and anti-chicken Alexa Fluor 647 (Thermo Fisher, Waltham, MA) diluted at 1:500 for 2 h at room temperature. Sections were then rinsed in PBST followed by addition to ddH₂0 and subjected to a Hoechst stain (Invitrogen, Carlsbad, CA) for 10 min.

All data generated or analyzed during this study are included in this article and its Online Resource.

Based on the success of the cos.Tet and MoPrP.Xho vectors for the generation of Tg mice [1, 15, 20, 22], we compared the Prnp genomic sequence of the rat with that of the mouse and Syrian hamster to identify conserved DNA sequences. We analyzed sequences with a scanning window of 100 bp for an identity greater than 50%. We identified the following regions: a sequence spanning a 6-kb upstream fragment, Exon 1, Intron 1, and Exon 2 (Region I); Intron 2 (Region II); and Exon 3/3’UTR and a 2.3 downstream sequence containing putative polyadenylation signals (Region III) (Fig. 1a). Because rats and mice only diverged ~10 million years ago [18], their sequences show a high degree of similarity. The largest difference occurs in Region I, which contains two indels of 0.6 and 1.1 kb (Fig. 1a). Rats diverged from hamsters ~25 million years ago [18], and in addition to a 1.1 kb indel in Region I, there is a 4.5 kb indel in Region II. Thus, we created a ~13.3 kb vector combining Regions I and III, the most highly conserved genetic elements of the rat Prnp locus.

We PCR amplified rat Prnp Region I and III fragments to contain overlapping 15 bp homology arms for In-Fusion cloning to each other and a pUC19 backbone. The endogenous Prnp ORF was removed from Region III and replaced by an XhoI site for cloning of genetic cargo, while NotI sites were added to remove the transgene from the pUC19 backbone (Fig. 1b). Finally, an ~13.3 kb construct was assembled by an In-Fusion reaction containing pUC19, Region I, and Region III fragments. Hereafter, we refer to this construct as the RaPrnp vector (Fig. 1b). The complete sequence of this vector is located in the Online Resource, Additional file 1: Supplementary Information 1.

To determine if RaPrnp drives gene expression in vivo, we cloned in a dual reporter cassette containing the LacZ gene followed by a T2A ribosomal skipping sequence and an EGFP ORF (Fig. 2a) into the XhoI site of RaPrnp to generate a RaPrnp-LacZ/EGFP construct. We microinjected this construct into SD rat zygotes. Approximately 65% of microinjected eggs survived, and ~10% of the eggs implanted into surrogate dams yielded live births (Table 1). From 13 live births, we obtained two founders that stably transmitted the RaPrnp-LacZ/EGFP transgene to their progeny (Table 1). These two independent founder lines, Tg12084 and Tg12085, were backcrossed to WT SD rats. Tg12084 rat brains displayed high EGFP fluorescence in young adult animals at 2 months of age compared with non-Tg animals, which lacked EGFP fluorescence (Fig. 2b–d). In addition to these findings, we transfected the RaPrnp-LacZ/EGFP construct into various rodent cell lines: rat neuroblastoma (B35), pheochromocytoma (PC12), rat embryonic fibroblast (RAT2), mouse neuroblastoma (N2a), catecholaminergic (CAD5), and mouse embryonic fibroblast (3T3) (Fig. 2e–j). Two days post-transfection, we observed strong EGFP fluorescence in the cytoplasm and moderate fluorescence in the neurite-like extensions in B35, PC12, N2a, and CAD5 cells (Fig. 2e, f, h, and i). Conversely, in the non-neuronal cell lines, we found a few modestly EGFP-positive RAT2 cells, while the transfected 3T3 cell line did not reveal any EGFP fluorescence (Fig. 2g and j). Because RaPrnp was capable of facilitating gene expression in mouse neuronal cell lines (Fig. 2h and i), we determined that murine regulatory elements of Prnp were preserved in this vector.

Having observed expression in 2-month-old RaPrnp-LacZ/EGFP Tg rats, we sought to determine if the RaPrnp vector supported gene expression during embryogenesis and at early postnatal stages. For the purpose of staging during development and birth, we refer to the day a mating plug is observed as embryonic day 0.5 (E0.5), while the day of birth for neonatal pups is indicated as postnatal day 0 (P0), respectively. At the E9.5 time point, EGFP fluorescence was not apparent in either line; however, at the E13.5 time point, the Tg12084 line showed in vivo EGFP fluorescence in the mesencephalon, telencephalon, and eye (Fig. 3a). Additionally, in these animals, EGFP fluorescence continued to expand globally in the brain and spinal cord at E18.5, P0, and P10 (Fig. 3b–e). In contrast to the Tg12084 line, we did not observe EGFP fluorescence in the CNS of Tg12085 rats until P10 (Fig. 3f).

Next, we studied LacZ and EGFP expression in adult Tg rats up to one year of age. We observed high EGFP fluorescence in the brains of 9-month-old Tg12084 and Tg12085 rats (Fig. 4a and b). Both lines demonstrated EGFP fluorescence in the cortex, corpus callosum, cerebellum, and brainstem (Fig. 4a and b). Tg12085 rats showed more widespread EGFP fluorescence that was higher in intensity in the brainstem and thalamic structures compared with Tg12084 animals. Conversely, Tg12084 rats had high EGFP fluorescence in the corpus callosum and forebrain (Fig. 4a). This region-specific EGFP fluorescence was maintained in one-year-old Tg12084 and Tg12085 animals (Fig. 4c and d). Tg12084 rats displayed higher EGFP fluorescence in the cortex, corpus callosum, and hippocampus (Fig. 4c). In contrast to the Tg12084 line, coronally sliced Tg12085 rat brain showed higher EGFP fluorescence in the thalamus and midbrain structures (Fig. 4d). To visualize the extent of RaPrnp-mediated expression in neural anatomical structures, we stained coronal sections of Tg12084 brain tissue with X-gal to measure LacZ activity in cells. While layer I lacked LacZ activity, we observed LacZ activity in cortical layers II–VI, with the highest reporter activity in layers II and III (Fig. 5a, inset). Furthermore, the nuclei of the CA1 layer and dentate gyrus showed stronger LacZ activity than the CA3 layer of the hippocampus, while the thalamus displayed sparse activity (Fig. 5a).

To identify which CNS cell types were positive for RaPrnp-mediated expression, we performed immunofluorescence staining and confocal microscopy in Tg12084 rat brains to detect if neuronal or glia cell types expressed EGFP. Using this method, we observed EGFP to label cell bodies and axons of neurons that were also positive for the NeuN protein in the cortex (Fig. 5b–d). Furthermore, the hippocampus of Tg12084 rats showed widespread EGFP expression (Fig. 5e), whereas EGFP and NeuN were highly co-localized in neurons of the CA1 layer (Fig. 5l). Although EGFP expression in the CA3 layer was greatly reduced (Fig. 5g), it was apparent in neurons (Fig. 5m). Notably, in both regions, EGFP did not label all neurons but instead yielded mosaicism in neuronal populations. EGFP did not co-localize with either Iba1 or GFAP positive microglia or astrocytes, respectively, in the cortex (Fig. 6a, d, g, and j) or the hippocampal CA1 (Fig. 6b, e, h, and k) and CA3 (Fig. 6c, f, i, and l) layers, suggesting that RaPrnp-mediated expression primarily targets neuronal cell types.

We and others have shown that mice overexpressing mouse PrP^C have shorter incubation periods compared with WT mice infected with RML prions [2, 5, 23]. In addition, we demonstrated in a number of models that transgene expression level is inversely correlated to susceptibility to disease onset [15, 20, 22]. We therefore posited that genetically expressing higher levels of PrP^C would provide more substrate for PrP^Sc conversion and an accelerated prion disease phenotype in rats infected with rat-passaged RML (rat RML) prions.

To overexpress rat PrP^C in the CNS, we PCR amplified the rat Prnp ORF with 15 bp homology arms and targeted insertion by In-Fusion cloning into an XhoI digested RaPrnp vector (Fig. 7a). This construct was microinjected into SD rat zygotes, and we achieved 81% viability, with 13% of implanted zygotes yielding live births (Table 1). Out of the 64 pups, we identified 15 potential founders (Table 1) by PCR genotyping. To determine whether transgene copy number was correlated with transgene expression level [19], we performed droplet digital PCR (ddPCR) using primer and probe sets targeting Exon I of the rat Prnp gene and western blotting to evaluate PrP protein levels (Fig. 7b and c; Online Resource, Additional file 1: Figure S1). Using this method, we detected copy numbers as low as 4 RaPrnp-PrP copies and a maximum of 132 copies in 12 Tg(RaPrnp-PrP) rats (Fig. 7b and c). For copy numbers greater than 10, we observed no correlation with RaPrnp-PrP copy number and PrP expression levels in the brain. In contrast, we observed a strong correlation with fewer than 10 RaPrnp-PrP copies on PrP expression levels (R² = 0.70; Fig. 7c). Of the seven potential founders paired with WT rats, three did not produce Tg offspring, while two lines were poor breeders. For this study, we used two founder lines, Tg2919 and Tg2922, which had 7 and 71 RaPrnp-PrP copies, respectively (Fig. 7b and c). Tg2919 and Tg2922 lines had total PrP^C expression levels 4.4× and 9.7× of WT animals (Online Resource, Additional file 1: Figure S1).

To evaluate transgene-mediated tissue specificity, we performed western blotting and probed for total PrP expression in brain, lung, liver, kidney, muscle, and heart tissue extracts from WT, Tg2919, and Tg2922 rats (Fig. 7d). While we consistently found higher expression of glycosylated PrP in the range of 30–40 kDa in brain extracts from Tg2919 and Tg2922 rats compared with WT animals, we also observed a faint endogenous band around 30 kDa in lung, liver, kidney, and heart extracts with similar levels of expression in WT and Tg animals (Fig. 7d). The observation of a prominent ~15 kDa band was detectable in most tissues and was similarly expressed in WT and Tg animals (Fig. 7d).

To observe normal prion disease progression in WT rats, we inoculated rat RML prions into WT animals, which reached disease onset at 175 ± 3 days post inoculation (dpi) (Fig. 7e and Table 2). To test the effect of additional PrP in Tg rats on prion disease kinetics, we inoculated 2-month-old Tg2919 rats with rat RML prions. The animals reached disease onset at 149 ± 2 dpi (Fig. 7e and Table 2). To determine if we could further accelerate prion disease in rats, we inoculated Tg2922 animals, which express more than twice the level of PrP than Tg2919 rats. Rat RML-infected Tg2922 rats succumbed to rapid disease onset at 112 ± 0 dpi (Fig. 7e and Table 2). Interestingly, terminal Tg2922 rats had lower levels of PrP^Sc compared with rat RML-infected WT and Tg2919 animals (Fig. 7f–h).

H&E staining was performed on WT, Tg2919, and Tg2922 RML-infected rat brain and compared with age-matched Tg control animals and WT rats inoculated with normal brain homogenate (NBH). Minimal vacuolization was observed in the brains of NBH-inoculated WT rats and age-matched uninoculated Tg2919 and Tg2922 controls (Fig. 8a, e, and i). In contrast, in both rat RML-infected WT and Tg2919 rats, frequent vacuolization was found within the hippocampus, striatum, and motor and sensory cortices (striatum depicted in Fig. 8b and f; Online Resource, Additional file 1: Figure S2). Conversely, Tg2922 rat brain showed the highest vacuolization within the brainstem (Fig. 8j), hypothalamus, and thalamus (Online Resource, Additional file 1: Figure S2). Hydrocephalus was apparent in all of the rats inoculated with rat RML and was most appreciable in the anterior horn of the lateral ventricles.

With respect to PrP immunofluorescence staining, all WT rats inoculated with NBH lacked PrP immunoreactivity following hydrolytic autoclaving (Fig. 8c). Notably, uninoculated Tg2919 animals occasionally had diffuse PrP staining in the striatum (Fig. 8g), while Tg2922 rats had some focal PrP immunoreactivity within the brainstem (Fig. 8k). In addition, both lines had some PrP immunoreactivity in the cingulate gyrus and hippocampus, which was likely a result of overexpression. WT and Tg2919 rats inoculated with rat RML prions had abundant PrP immunoreactivity, showing PrP deposits across numerous brain regions including the brainstem, cerebellum, hippocampus, thalamus, striatum, and motor and sensory cortices (representative photos of the striatum in Fig. 8d and h). PrP deposits in rat RML-infected Tg2922 rats were most severe within the brainstem (Fig. 8l) but were also present in the cerebellum, sensory cortices, and thalamus. No appreciable PrP deposits were noted in the motor cortices or striatum. Brains of prion-infected mice and rats are characterized by astrocytic gliosis; however, Tg2919 and Tg2922 rat lines had a high basal level of astrocytic gliosis, and, therefore, no upregulation was apparent in prion-infected rats.