Jiangsu Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 211166, China
b
Key Laboratory of Targeted Intervention of Cardiovascular Disease, Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Nanjing Medical University, Nanjing 211166, China
c
Department of Cell Biology, Guangdong Provincial Key Laboratory of Bone and Joint Degenerative Diseases, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
d
State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 211166, China
e
Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University, Shenzhen 518035, China
Tuberous sclerosis complex (TSC) is a dominant genetic neurocutaneous syndrome characterized by multiple organ hamartomas. Although rodent models bearing a germline mutation in either TSC1 or TSC2 gene have been generated, they do not develop pathogenic lesions matching those seen in patients with TSC because of the significant differences between mice and humans, highlighting the need for an improved large animal model of TSC. Here, we successfully generate monoallelic TSC1-modified Bama miniature pigs using the CRISPR/Cas9 system along with somatic cell nuclear transfer (SCNT) technology. The expression of phosphorylated target ribosomal protein S6 is significantly enhanced in the piglets, indicating that disruption of a TSC1 allele activate the mechanistic target of rapamycin (mTOR) signaling pathway. Notably, differing from the mouse TSC models reported previously, the TSC1 Bama miniature pig developed cardiac rhabdomyoma and subependymal nodules, resembling the major clinical features that occur in patients with TSC. These TSC1 Bama miniature pigs could serve as valuable large animal models for further elucidation of the pathogenesis of TSC and the development of therapeutic strategies for TSC disease.
Tuberous sclerosis complex (TSC) is a dominant hereditary disease characterized by a form of hamartoma and benign tumors involving multiple organs and systems (Islam and Roach, 2015). TSC shows highly spontaneous mutagenicity with an incidence of 1:6,000–1:10,000 worldwide, seriously affecting the life quality of patients in an age-related and sex-dependent manner (DiMario et al., 2015; Salussolia et al., 2019). The devastating complications caused by pathological changes in the nervous system, heart, kidney, and lungs lead to poor prognosis and even death. Although there are currently accepted diagnostic standards for TSC (Krueger and Northrup, 2013; Portocarrero et al., 2018), the detailed molecular pathogenesis of TSC remains to be elucidated for effective therapeutic purposes.
TSC is closely related to two tumor suppressor genes, TSC1 and TSC2 (Yu et al., 2014; Curatolo et al., 2015). Hamartin and tuberin proteins, encoded by TSC1 and TSC2 genes, respectively, form a heterodimer complex playing a crucial role in regulating cell growth, differentiation, and proliferation through the inhibition of the mechanistic target of rapamycin (mTOR) pathway (Cao et al., 2017; Saxton and Sabatini, 2017). Mutations in either TSC1 or TSC2 promote an overreaction of the mTOR pathway, leading to the occurrence of TSC (Lam et al., 2017; Randle, 2017). Numerous pathogenic mutations of TSC1 and TSC2 genes have been identified in patients, which not only enrich the TSC1 and TSC2 gene mutation spectrum but also show the genetic heterogeneity of TSC (Au et al., 2007; Martin et al., 2017). While the TSC2 mutations exhibit an arbitrary distribution, the TSC1 mutations are frequently reported in the 15th and 17th exons, indicating the existence of mutation hot spots in the TSC1 gene (van Slegtenhorst et al., 1999; Rubilar et al., 2017).
By conventional and conditional knockout of the TSC1 or TSC2 gene, researchers have successfully established and characterized various genetically modified mouse models of TSC (Kobayashi et al., 1999; Onda et al., 1999; Kobayashi et al., 2001; Kwiatkowski et al., 2002). These mouse models have been extensively used and provide valuable insights into the understanding of the pathogenesis and treatment of TSC. However, no mouse model is close to replicate the clinical features, such as cardiac rhabdomyosarcoma (CRM) and subependymal nodules, observed in patients with TSC, which in turn compromise the findings from these mouse models (Kwiatkowski, 2010). The significant differences in physiology between rodents and humans highlight the demand for the study of TSC using larger animal models closer to humans. Currently, pigs are extensively used in biomedical studies in that they have many similarities to humans concerning anatomy, physiology, and clinical relevance (Hai et al., 2017; Yao et al., 2019). More importantly, the CRISPR/Cas9 system along with somatic cell nuclear transfer (SCNT) technology enables the rapid generation of a variety of genetically engineered pigs mimicking human diseases (Chen et al., 2015; Yan et al., 2018). In addition, the short duration of pregnancy, large litter size, and ethical and economic acceptance make pigs a more preferable model than non-human primates for human disease research.
However, no pig model carrying the germline TSC1 mutation, which could be essential to dissect the molecular pathological mechanism of TSC, is yet available. Homozygosity of TSC1 mutation causes embryonic lethality in mice (Kobayashi et al., 2001), indicating that the function of hamartin is essential for mammalian development. Therefore, in the present study, we aim to generate monoallelic TSC1-modified pigs by CRISPR/Cas9-mediated gene targeting and initially characterize their phenotypes.
2.
Results
2.1
Generation of monoallelic TSC1-modified pigs using CRISPR/Cas9 technology
The TSC1 targeting sgRNA was designed using online tools (https://zlab.bio/guide-design-resources) based on the genomic sequence of the 15th exon of TSC1 of Bama miniature pigs determined by Sanger sequencing. The precise target site is shown in Fig. 1A. To establish TSC1-modified Bama miniature pig fetal fibroblast (PFF) cell lines, we simultaneously transfected a plasmid encoding the Cas9 endonuclease and sgRNA for TSC1 and a neomycin-resistant plasmid (pCMV-tdTomato) into early passages of PFFs derived from a male Bama miniature pig fetus. A total of 40 resistant colonies were harvested after G418 screening for ten days. PCR amplicons spanning the TSC1 target region were ligated into T-vectors for Sanger sequencing. As the homozygous TSC1 mutations caused embryonic lethality in mice (Kobayashi et al., 2001), we screened the TSC1-modified cell colonies that contained a wild-type (WT) TSC1 allele. Of the 40 colonies analyzed, eight were identified as heterozygous ones as each harbored a WT TSC1 allele and a modified TSC1 allele. These cell colonies were pooled and used as cell donors for the SCNT procedure. Twelve live-born male piglets were generated from two recipients (Fig. 1B). The genotypes of cloned piglets were analyzed by PCR sequencing using DNA isolated from their ear tissues. The results revealed that all cloned piglets, except #4 and #9, were heterozygous TSC1 mutants (Fig. 1C). Piglets #4 and #9 were identified as WT, probably owing to contamination of donor cells with a few WT PFFs, which were used as controls in this study.
Fig.
1.
Generation of monoallelic TSC1-targeted piglets. A: Schematic diagram of sgRNA targeting the TSC1 gene locus. The chromatogram of TSC1 15th exon sequences of WT Bama miniature pigs is partially shown, and the targeting site is noted. B: Photograph of newborn cloned piglets. C: Genotypes of cloned piglets. The WT TSC1 sequence is shown at the top. The sgRNA sequence is highlighted in blue, and the PAM sequence is highlighted in green and underlined. Deletions are indicated with (Δ). Italic red letters denote inserted base pairs.
Albeit the CRISPR/Cas9 system shows high efficiency in pig gene targeting, it might trigger DNA cleavage at undesired sites showing homology to the sgRNA sequence. Therefore, we screened the pig genome sequence using an online tool (http://www.rgenome.net/cas-offinder/) to evaluate the potential off-target sites (OTSs) of the TSC1 sgRNA. In this way, a total of 33 OTSs were computationally identified. We randomly chose 20 OTSs (Table S1) for experimental validation. The PCR amplicons spanning the OTSs from TSC1+/− piglets and WT controls were Sanger sequenced and aligned. No indel mutation was detected in the sequencing reads, indicating that the TSC1+/− piglets are unlikely to have off-target mutagenesis at these sites.
2.2
Hyperactivation of the mTORC1 signaling pathway in TSC1 pigs
TSC1 gene variations result in overreaction of the mTOR pathway and lead to occurrence of TSC (Lam et al., 2017; Randle, 2017). Among the analyzed samples, TSC1+/− piglet #5 died shortly after birth but showed no external developmental abnormalities in weight, body shape, and skin appearance. To determine whether mTOR signaling was altered in piglet #5, we first conducted a quantitative reverse transcription PCR (qRT-PCR) analysis to assess TSC1 gene expression in the heart and kidney tissues because no commercial antibody against porcine TSC1 is available. Piglet #9 (WT) was used as control. As expected, the TSC1 gene was significantly downregulated in both the TSC1+/− piglet heart and kidney compared with the WT control (Fig. 2A). We then analyzed mTORC1 activity in porcine hearts and kidneys using a readout of the phosphorylated target ribosomal protein S6, a direct substrate of mTORC1 (Ben-Sahra and Manning, 2017). TSC1+/− piglet #5 displayed a significantly higher ratio of phosphor-S6/S6 than piglet #9, namely, a 4- to 5-fold increase relative to the WT piglet, indicating the hyperactivation of mTORC1 signaling due to the monoallelic modification of TSC1 (Fig. 2B). Consistent with the Western blot results, p-S6 expression was significantly elevated in the heart and kidney tissues of the TSC1+/− piglet compared with the WT piglet, as revealed by immunohistochemistry (IHC) staining (Fig. 2C). To examine whether hyperactivation of mTORC1 promotes excessive cell growth and proliferation, pig spleen paraffin sections were subjected to Ki67 staining. The results indicated that the proliferation of spleen cells was significantly enhanced in the TSC1+/− neonatal piglet compared with the WT control (Fig. 2D).
Fig.
2.
Hyperactivation of the mTORC1 signaling pathway in the neonatal TSC1 piglet. A: Quantitative reverse transcription PCR analysis of TSC1 gene expression levels in the heart and kidney tissues of TSC1 neonatal piglet #5 and age-matched WT control piglet #9. B and C: The expression of phosphorylated ribosomal target protein S6 is significantly increased in the heart and kidney tissues of theTSC1 neonatal piglet compared with the WT piglet revealed by Western blot (B) and immunohistochemistry staining (brown staining). D: Representative images of Ki67 staining of splenocytes of the neonatal TSC1 piglet and age-matched WT pig (left). The Ki67-positive splenocytes were counted under randomly selected five non-overlapping microscope fields (right). Results are presented as mean ± SD. ∗∗∗, P < 0.001. Scale bars, 20 μm (C), 50 μm (D). SD, standard deviation.
We further assessed TSC1 and p-S6 protein expression in the heart and kidney tissues derived from #3, #7, and #8 adult TSC1+/− pigs. Pig #4 was used as age-matched WT control. The results of qRT-PCR, Western blot, and IHC showed that the TSC1 gene was downregulated and p-S6 was significantly enhanced in the TSC1+/− pigs compared with the WT pig, suggesting that mTOR signaling is sustainably activated upon the absence of a TSC1 allele (Fig. 3A–C). Ki67 staining showed that adult TSC1+/− pigs had appreciable proliferation spleen cells compared with the age-matched WT control (Fig. 3D). Together, these results indicate that the heterozygous TSC1 mutation contributes to the overreaction of the mTOR signaling pathway in pigs.
Fig.
3.
The mTORC1 signaling pathway is hyperactivated in adult TSC1 pigs. A: Quantitative reverse transcription PCR analysis of TSC1 gene expression levels in the heart and kidney tissues of adultTSC1 piglets (#3, #7, and #8) and the age-matched WT control (#4). B and C: The expression of phosphorylated ribosomal protein S6 in the heart and kidney of TSC1 pigs and WT control was examined by Western blot (B) and immunohistochemistry staining (brown staining). D: Representative images of Ki67 staining of splenocytes of the adult TSC1 piglet and age-matched WT control (left). The Ki67-positive splenocytes were counted under randomly selected five nonoverlapping microscope fields (right). Results are presented as mean ± SD. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗,P < 0.001. Scale bars, 100 μm (C), 50 μm (D). SD, standard deviation.
2.3
Development of brain nodular lesions in the heterozygous TSC1-mutant neonatal piglet
Pathological changes in the central nervous system, including cortical or subcortical and subependymal nodules, can lead to severe complications and even death in patients with TSC (Lu et al., 2018). To determine whether losing a TSC1 allele causes neurological system damage in pigs, we examined the brain of piglet #5 and used piglet #9 as the WT control. No nodule could be seen on the brain surface of either the TSC1-mutant or the WT piglet. However, compared with piglet #9, the brain of piglet #5 showed severer edema, wider gyrus, shallower sulci, and more transparent meninges on the brain surface (Fig. 4A and B), which are similar to the clinical features of patients with TSC. The brains of piglets #5 and #9 were further examined by using magnetic resonance imaging (MRI) T2-weighted imaging. High-density shadows varying in size were seen in the lateral ventricles on both sides of theTSC1+/− newborn piglet brain with asymmetrical distribution but not in the control (Fig. 4C and D). Moreover, there are many irregularly distributed shadows in the cerebellum and medulla oblongata of the TSC1+/− piglet (Fig. 4E–h). We inferred that the TSC1+/− newborn piglet developed subependymal nodules in the brain owing to the lack of one of the two WT TSC1 alleles.
Fig.
4.
Brain nodules in the TSC1 neonatal piglet. A: Macroscopic appearance of the TSC1 (#5) and WT (#9) newborn piglets whole brains. B: Macroscopic sagittal view of the TSC1 and WT neonatal pig brains. Compared with the WT control, the TSC1 piglet exhibits visible edema, wider dorsal brain, shallower sulci, and transparent meninges surface. C–H: Representative MRI T2-weighted images of the TSC1 and WT neonatal piglet brains. c–h: High magnifications of the area enclosed by a rectangle in the left panel. White arrows denote the nodules in the ventricle, cerebellum, and medulla oblongata of TSC1 neonatal pig brains. Scale bars, 1 cm (C–H), 0.5 cm (c, d), 0.25 cm (e–h). MRI, magnetic resonance imaging.
Cardiac rhabdomyoma occurs in 47–67% of patients with TSC, usually with the most massive tumor volume in the neonatal period and which shrinks to disappear with age (Shen et al., 2015; Aw et al., 2017). Therefore, heart tissue sections of piglets #5 and #9 were examined by hematoxylin and eosin (H&E) staining to assess the presence of cardiac rhabdomyoma. Unlike the WT control, theTSC1+/− newborn piglet heart tissue exhibited lesions with a clear boundary formed by hyperplasia of large vacuolar cells. The nodular cells displayed diverse morphologies and abundant eosinophilic cytoplasm (Fig. 5A). Compared with the adjacent cells, the nodular cells showed significantly increased glycogen content, as indicated by periodic acid-Schiff (PAS) staining (Fig. 5B). IHC analysis revealed that the striated muscle markers, including myoglobin, desmin, and vimentin, were strongly expressed in the nodular cells (Fig. 5C). However, Ki67 was barely detected in the nodular cells, indicating that these cells had no mitotic activity, and the lesions were more likely to be hamartomas than neoplasms (Fig. 5C). Taken together, we inferred that the monoallelic mutation in the TSC1 gene led to TSC in pigs as they developed subependymal nodules and cardiac rhabdomyoma, which are the two major clinical manifestations used to diagnose patients with TSC. Notably, the neonatal TSC1+/− newborn piglet did not develop any renal lesions, liver lesions, or epilepsy seen in TSC1+/− mice. Moreover, we did not observe pathological manifestations in the remaining TSC1+/− piglets even when they were at 16 months of age, partly owing to the genetic heterogeneity of TSC.
Fig.
5.
Cardiac rhabdomyoma in the TSC1 piglet. A: H&E staining shows cardiac rhabdomyoma in the heart tissue ofTSC1 neonatal piglet #5. The dashed line circumscribes the boundary of the tumor. B: Representative image of PAS staining of cardiac rhabdomyosarcoma in the heart tissue of TSC1 neonatal piglet #5. C: Representative immunohistochemistry images of cardiac rhabdomyosarcoma in the heart tissue of TSC1 neonatal piglet #5. Scale bars, 100 μm (A and B); 200 μm (C). H&E, hematoxylin and eosin; PAS, periodic acid-Schiff.
Animal models are valuable tools to elucidate the pathological mechanism of TSC and the development of highly specific and efficacious treatments. However, the most widely used mouse models bearing mutations in an allele of TSC1 or TSC2 have limitations as they do not sufficiently recapitulate the pathologic lesions seen in patients with TSC (Kobayashi et al., 2001; Kwiatkowski, 2010; Guo et al., 2016). Pigs have advantages over rodents in modeling human diseases because they have more similarities with humans in anatomy, physiology, and clinical relevance. However, the utilization of genetically engineered pigs as TSC models is still a vacancy. Here, we successfully establish Bama miniature pigs with monoallelic modification of TSC1 using the CRISPR/Cas9 system combined with SCNT technology. The mTORC1 signaling pathway is hyperactivated in both neonatal and adult TSC1+/− Bama miniature pigs. Moreover, the neonatal TSC1+/− Bama miniature pig exhibits CRM and subependymal nodules, which are the two major clinical features for TSC diagnostic criteria, indicating that the loss of aTSC1 allele leads to the occurrence of TSC in pigs.
Mutations in either TSC1 or TSC2 gene cause similar disease phenotypes by disrupting the interaction between their encoded proteins, hamartin and tuberin. Numerous mutations scattered in the genomic regions of these two genes have been extensively associated with TSC phenotypes. A large proportion of TSC1 gene mutations are detected in exons 15 and 17, whereas unanimous hot spot mutations in the TSC2 gene are yet to be determined. Most TSC1 variations are small insertions or deletions, which are also the most common modification forms induced by the CRISPR/Cas9 system via its preference for the error-prone nonhomologous end joining mechanism. The CRISPR/Cas9 technology has been widely applied in pig gene targeting because of its high mutagenesis efficiency. Therefore, the CRISPR/Cas9 system was used to target the 15th exon of TSC1 in this study. Consistent with previous studies (Fang et al., 2018), the CRISPR/Cas9 system showed high gene targeting efficiency in PFFs as eight monoallelic TSC1-modified colonies were obtained in one transfection experiment. A total of 12 TSC1+/− piglets with three kinds of indels in the targeted region were produced by just one round of SCNT, supporting that the CRISPR/Cas9 system in combination with SCNT can significantly facilitate the efficient generation of gene-modified pigs.
The ablation of TSC1 in the brain affects cellular differentiation, migration, and proliferation of the central nervous system, causing characteristic brain lesions in patients. CRM is frequently diagnosed in infants and children associated with TSC1 mutations (Uzun et al., 2007). Our TSC1+/− neonatal piglets showed CRM clearly on histological sections and noncalcified nodules in the subependymal region, medulla oblongata, and cerebellum as per MRI scanning. These phenotypes caused by TSC1 mutations in pigs resemble those in human patients with TSC, reflecting that the function of hamartin is highly conserved in both species. Unfortunately, the TSC1+/− neonatal piglet was in poor overall condition and died soon after birth for uncertain reasons. We suspected that CRM located on the ventricular muscle and/or the widespread subependymal nodules in the brain was a possible cause of its death. We also noticed that the TSC1+/− newborn piglets had an edematous brain surface, widened dorsal brain, and shallow sulci, indicating that the TSC1 gene affects brain development. Further investigation is required. No clinical symptoms associated with patients with TSC were seen in the remaining genetically modified TSC1 pigs even at the age of 16 months, partly because of the genetic heterogeneity of TSC, which was well documented (Martin et al., 2017). As TSC2+/− mice showed more severity in tumor development than TSC1+/− mice, we speculated that the establishment of TSC2-modified pigs would be a choice in future studies to obtain TSC animal models with apparent manifestations. Surprisingly, although the mTOR signal was sustainably upregulated in the TSC1+/− piglets, we have not seen any renal lesions, liver lesions, or epilepsy, which were present in TSC1-knockout mouse models. These discrepancies may raise some crucial issues in terms of pathology and phenotype differences between mice and large animal models of TSC disease.
Our study demonstrates that pig models can more faithfully recapitulate clinical features observed in patients with TSC, indicating that pigs are a valuable tool to model TSC and develop therapeutic strategies. However, there are some limitations in using large animal models because of the great technical difficulty, the expensive animal facility, and the limited sample size. While the sex-dependent difference in TSC incidence has been confirmed in humans and mouse models, we did not consider gender effects on phenotype as only male TSC1+/− piglets were produced in this study. Moreover, a more extensive survey is required because it may take longer than 16 months for the TSC1+/− pigs to develop TSC lesions.
In conclusion, monoallelic TSC1-modified Bama miniature pigs exhibited characteristic TSC features, including CRM and subependymal nodules, which were not observed in TSC1- or TSC2-knockout mouse models. Our TSC1-knockout pigs will serve as useful preclinical animal models to elucidate the pathogenic mechanism of TSC and develop clinical diagnosis and effective treatment of TSC.
4.
Materials and methods
4.1
Animals
Both WT adult Bama miniature pigs and Landrace pigs were purchased from Zhengda Co., Ltd. (Huaiyin, China) and raised in a large animal facility affiliated to Nanjing Medical University. Standard feeding procedures were applied to all animals. Isoflurane anesthesia was used in euthanasia and embryo transplantation to minimize the suffering of pigs. We conducted all animal experiments in accordance with the requirements approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
4.2
CRISPR/Cas9 plasmid constructs
sgRNA for the porcine TSC1 gene was designed using online software (https://zlab.bio/guide-design-resources) and synthesized by GenScript (Nanjing, China) (TSC1 sgRNA:5′-CACCGTACGACCACCTTTTTGAGG-3′, 5′-AAACCCTCAAAAAGGTGGTCGTAC-3′). These oligos were mixed and incubated at 37°C for 30 min, heated at 95°C for 5 min, and then cooled down to 25°C at a rate of 5°C/min. The annealed oligos were then subcloned into Bbs I (Thermo Fisher Scientific, Waltham, USA) digested pX330 plasmid to generate the TSC1-targeting vector.
4.3
PFF transfection and selection
Approximately, 1.5 × 106 early passages of Bama miniature pig PFFs were suspended in 100 μL of Nucleofector solution (Amaxa Biosystems/Lonza, Cologne, Germany) with 5 μg of TSC1 Cas9-sgRNA targeting plasmid and 1 μg of the neomycin-resistant plasmid (pCMV-tdTomato). Electroporation was performed using nuclear infection procedure u-023 following the manufacturer's instructions. Two days later, G418 (Gibco, Grand Island, USA) was added to the post-transfection cells at a concentration of 800 μg/mL and maintained for ten days thereafter. The G418-resistant single-cell colonies were picked and inoculated into 24-well plates. When a confluence of 80% or more was reached, colonies were passaged to 12-well plates. Partial cells of each cell colony were used for PCR genotyping, and the rest of the cells were used as donors for SCNT. The primers used for the TSC1-targeted region amplification were as follows: forward: 5′-TTCGACTCTCCCTTCTACCG-3′; reverse: 5′-ATTAGCCTGTCCAGCACCTC-3′. PCR was performed with standard protocols as described previously by Chen et al. (2018). The PCR amplicons were ligated into a pMD18-T vector (Takara Clontech, Tokyo, Japan). A total of 15–20 individual clones for each amplicon were subjected to Sanger sequencing.
4.4
SCNT and embryo transfer
The oocytes were obtained from ovaries of 6-month gilts, which were collected and cultured for maturation (approximately 42–44 h). The enucleation of the mature oocytes was conducted as previously described by Zhang et al. (2018). A single donor cell was injected into the perivitelline cytoplasm of an enucleated oocyte. After electrofusion, the reconstructed embryos were cultured at 38.5°C for 24 h. A total of 250–300 reconstructed embryos were used for embryo transplantation. Pregnancy examination and delivery procedures were performed as described previously by Fang et al. (2018).
4.5
Genotyping and qRT-PCR analysis
The ear tissues of newborn piglets were used to isolate genomic DNA using the QIAamp DNA Mini Kit (QIAGEN, Dusseldorf, Germany). PCR procedures described previously by Chen et al. (2018) were used to genotype the cloned piglets using primers for the TSC1 gene (forward: 5′-TTCGACTCTCCCTTCTACCG-3′, reverse: 5′-ATTAGCCTGTCCAGCACCTC-3′). Total RNA was isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions and reverse transcribed into cDNA using the HiScript®II QRT SuperMix (Vazyme, Nanjing, China). The target genes were amplified in three replicates using the SYBR Green PCR Master Mix (Vazyme, Nanjing, China). The primers specific for porcine TSC1 were 5′-TAAAGGGCGGCAGTGAATGT-3′ and 5′-GCATTTGCTTCTAGGCGCTC-3′. The porcine GAPDH gene (forward: 5′-GCAAAGTGGACATTGTCGCCATCA-3′; reverse: 5′-TCCTGGAAGATGGTGATGGCCTTT-3′) was used as an internal control. The qRT-PCR procedures were as follows: 95°C for 5 s, followed by 40 cycles at 95°C for 10 s and 60°C for 30 s. We used the melting curve analysis to evaluate the amplification specificity of qRT-PCR and used the 2−ΔΔCt method (Pfaffl, 2001) to measure the relative gene expression levels.
4.6
Histological analysis
Porcine tissues including the heart, kidney, and spleen derived from two WT (#4 and #9) and four TSC1+/− piglets (#3, #5, #7, #8) were fixed in 4% paraformaldehyde (PFA) over 24 h, dehydrated, and then embedded in paraffin blocks. The 5-μm sections were deparaffinized and rehydrated for H&E staining and PAS reaction as previously described byFu and Campbell-Thompson (2017) and Chen et al. (2018). For immunohistochemistry analysis, tissue sections were heated at 120°C for 3 min in 10 mM citric acid buffer (pH 6.0) to retrieve the antigen. To block endogenous peroxidase activity, tissue sections were rinsed with 3% H2O2 in methanol for 10 min after washing in PBST three times, then cleaned with PBST, and subsequently blocked with 10% goat serum for 1 h at room temperature. Tissue sections were then incubated with diluted primary antibodies overnight at 4°C. The following primary antibodies were used: anti–phosphor-S6 (S235/236) (#2211; CST, Boston, MA, USA), anti-Ki67 (ab15580; Abcam, Cambridge, MA, USA), anti-desmin (BM4101; BOSTER, Wuhan, China), anti-myoglobin (DF7358; Affinity Biosciences, Wuhan, China), and anti-vimentin (GB14167; Servicebio, Wuhan, China). The tissue sections were incubated with a goat anti-rabbit secondary antibody (sc-2004; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature, then washed, and stained with diaminobenzidine/imidazole. Immunohistochemistry images were acquired using an Olympus FSX100 microscope (Olympus, Tokyo, Japan).
4.7
Western blot analysis
A total of 20–30 μg of heart or kidney tissue extract was used for Western blot analysis. Total proteins were separated by 12% SDS-PAGE gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA) by electroblotting. The membranes were incubated at 4°C overnight with anti-phosphor-S6 (S235/236) or anti-S6 (#2217; CST, Boston, MA, USA) primary antibody. Goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (dilution: 1:2000; CWBiotech, Shanghai, China) was incubated with the membranes at 25°C for 1 h. The SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) was used for protein detection. Images were captured using a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).
4.8
MRI analysis
The whole porcine brains derived from two WT (#4 and #9) and four TSC1+/− piglets (#3, #5, #7, #8) were taken out from the cranial cavity and then fixed in 4% PFA for more than 48 h. The Biospec 7T/20 USR magnetic resonance system (Burker, Germany) was used for coronal scanning. The scanning parameters were as follows: RARE sequence T2WI: TR 3679.2 ms, TE 33.0 ms; layer thickness, 1.5 mm; and layer gap, 1.5 mm.
4.9
Statistical methods
All data were presented as mean ± SD. Data from two groups were analyzed by a two-tailed unpaired Student's t-test. A P-value of 0.05 is set as the threshold value of statistical significance.
We thank all members of Jiangsu Key Laboratory of Xenotransplantation for their helpful suggestions regarding the manuscript. This work was supported by grants from the National Natural Science Foundation of China (31701283, 81970164), the National Key R&D Program of China (2017YFC1103701, 2017YFC1103702), the Jiangsu Key Laboratory of Xenotransplantation (BM2012116), the Sanming Project of Medicine in Shenzhen, the Fund for High Level Medical Discipline Construction of Shenzhen (2016031638), and the Shenzhen Foundation of Science and Technology (JCYJ20160229204849975, GCZX2015043017281705). The authors declared that they have no conflict of interest.
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Figure 1. Generation of monoallelic TSC1-targeted piglets. A: Schematic diagram of sgRNA targeting the TSC1 gene locus. The chromatogram of TSC1 15th exon sequences of WT Bama miniature pigs is partially shown, and the targeting site is noted. B: Photograph of newborn cloned piglets. C: Genotypes of cloned piglets. The WT TSC1 sequence is shown at the top. The sgRNA sequence is highlighted in blue, and the PAM sequence is highlighted in green and underlined. Deletions are indicated with (Δ). Italic red letters denote inserted base pairs.
Figure 2. Hyperactivation of the mTORC1 signaling pathway in the neonatal TSC1 piglet. A: Quantitative reverse transcription PCR analysis of TSC1 gene expression levels in the heart and kidney tissues of TSC1 neonatal piglet #5 and age-matched WT control piglet #9. B and C: The expression of phosphorylated ribosomal target protein S6 is significantly increased in the heart and kidney tissues of theTSC1 neonatal piglet compared with the WT piglet revealed by Western blot (B) and immunohistochemistry staining (brown staining). D: Representative images of Ki67 staining of splenocytes of the neonatal TSC1 piglet and age-matched WT pig (left). The Ki67-positive splenocytes were counted under randomly selected five non-overlapping microscope fields (right). Results are presented as mean ± SD. ∗∗∗, P < 0.001. Scale bars, 20 μm (C), 50 μm (D). SD, standard deviation.
Figure 3. The mTORC1 signaling pathway is hyperactivated in adult TSC1 pigs. A: Quantitative reverse transcription PCR analysis of TSC1 gene expression levels in the heart and kidney tissues of adultTSC1 piglets (#3, #7, and #8) and the age-matched WT control (#4). B and C: The expression of phosphorylated ribosomal protein S6 in the heart and kidney of TSC1 pigs and WT control was examined by Western blot (B) and immunohistochemistry staining (brown staining). D: Representative images of Ki67 staining of splenocytes of the adult TSC1 piglet and age-matched WT control (left). The Ki67-positive splenocytes were counted under randomly selected five nonoverlapping microscope fields (right). Results are presented as mean ± SD. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗,P < 0.001. Scale bars, 100 μm (C), 50 μm (D). SD, standard deviation.
Figure 4. Brain nodules in the TSC1 neonatal piglet. A: Macroscopic appearance of the TSC1 (#5) and WT (#9) newborn piglets whole brains. B: Macroscopic sagittal view of the TSC1 and WT neonatal pig brains. Compared with the WT control, the TSC1 piglet exhibits visible edema, wider dorsal brain, shallower sulci, and transparent meninges surface. C–H: Representative MRI T2-weighted images of the TSC1 and WT neonatal piglet brains. c–h: High magnifications of the area enclosed by a rectangle in the left panel. White arrows denote the nodules in the ventricle, cerebellum, and medulla oblongata of TSC1 neonatal pig brains. Scale bars, 1 cm (C–H), 0.5 cm (c, d), 0.25 cm (e–h). MRI, magnetic resonance imaging.
Figure 5. Cardiac rhabdomyoma in the TSC1 piglet. A: H&E staining shows cardiac rhabdomyoma in the heart tissue ofTSC1 neonatal piglet #5. The dashed line circumscribes the boundary of the tumor. B: Representative image of PAS staining of cardiac rhabdomyosarcoma in the heart tissue of TSC1 neonatal piglet #5. C: Representative immunohistochemistry images of cardiac rhabdomyosarcoma in the heart tissue of TSC1 neonatal piglet #5. Scale bars, 100 μm (A and B); 200 μm (C). H&E, hematoxylin and eosin; PAS, periodic acid-Schiff.
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