Transgenic Minipigs Research Articles

Characterization of hGFAP-DsRed Transgenic Guangxi Bama Mini-Pigs and Their Offspring

Characterization of hGFAP-DsRed Transgenic Guangxi Bama Mini-Pigs and Their Offspring

Upregulated expression of proteolytic enzymes in the cultured retinal pigment epithelial cells of minipig transgenic for the human mutated huntingtin

PurposeHuntington′s disease (HD) belongs to the hereditary neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the protein of huntingtin (Htt). Since mutant Htt (mtHtt) and especially their small proteolytic fragments are very toxic to all HD cells (particularly those of neuroectodermal origin such as neurons or retinal pigment epithelial cells), it has been suggested that upregulated proteolysis of mtHtt plays a crucial role in the HD pathogenesis including damage of the retina. Therefore, the purpose of the present study was to investigate the possible participation of the proteolytic enzymes from the group of caspases, matrix metalloproteinases (MMP), calpains in HD pathology of retinal pigment epithelial cells.MethodsIn this study we used wild type (WT) and transgenic minipigs for N‐terminal part of the human mtHtt (TgHD) (548aaHTT‐145Q, F2 generation age 36 and 48 months, respectively). Proteases were examined in cultured retinal pigment epithelial cells immunocytochemically (ICC) or biochemically by western blotting (WB) using the following primary antibodies: anti‐caspase‐3, anti‐caspase‐8, anti‐calpain‐5, anti‐MMP‐9.ResultsUsing biochemical and immunocytochemical analysis, we detected increased expressions of caspase‐3, caspase‐8, matrix metalloproteinase‐9, calpain‐5, and probably its multiple proteolytic cleavage products (generated from mtHtt) in cultured RPE cells of TgHD minipigs in comparison to WT animals.ConclusionsIncreased expression of proteolytic enzymes in TgHD RPE cells can contribute to the retinal damage during development of HD.

Emerging roles of IFNα, IL-10, IL-1 and IL-8 in central nervous inflammation and immune response imbalance in transgenic porcine model of neurodegenerative Huntington’s disease

Abstract Huntington’s disease (HD) is an inherited neurodegenerative disease with impairment of motor and cognitive functions. Studies in HD gene carriers expressing mutant huntingtin protein (mHTT) demonstrated altered immune response indicating that cytokines may have a significant role in disease development. We utilized the novel transgenic HD model bearing N-fragment of human mHTT in minipig (Baxa M et al., J Huntington’s Dis. 2013, 2:47–68), to investigate central nervous and peripheral cytokine levels. Using Luminex® xMAP™ multiplexing technology and Thermo Fisher Scientific Inc., Swine Cytokine Magnetic 7-plex Panel (Invitrogen™ LSC0001M), the biological fluids were interrogated to identify cytokine alterations related to HD progression. The modelling of interaction networks between such cytokines and HTT facilitated study of intracellular signaling pathways associated with immune response dysregulation. The most pronounced change was the decline of IFNα in cerebrospinal fluid and secretome of microglial cells (MG). In addition, IL-10 was also lower in the same biological fluids. Elevated levels of pro-inflammatory IL-1 and IL-8 were secreted by MG whilst IL-8 was also increased in serum of transgenic minipigs. The high proportion of mHTT in MG may have a causative impact on cytokine production. A novel role for CREB-binding protein in HD pathogenesis was highlighted. These findings underlined the roles of IFNα, IL-10, IL-1 and IL-8 in central nervous system inflammation and immune response imbalance in HD progression warrants further investigation into their usefulness in human patients. Acknowledgement We thank CHDI, MESY Projects EXAM; CZ.1.05/2.1.00/03.0124, LO1609 and Thermo Fisher Scientific for expertise.

Mutated Huntingtin Causes Testicular Pathology in Transgenic Minipig Boars

Background: Huntington's disease is induced by CAG expansion in a single gene coding the huntingtin protein. The mutated huntingtin (mtHtt) primarily causes degeneration of neurons in the brain, but it also affects peripheral tissues, including testes. Objective: We studied sperm and testes of transgenic boars expressing the N-terminal region of human mtHtt. Methods: In this study, measures of reproductive parameters and electron microscopy (EM) images of spermatozoa and testes of transgenic (TgHD) and wild-type (WT) boars of F1 (24-48 months old) and F2 (12-36 months old) generations were compared. In addition, immunofluorescence, immunohistochemistry, Western blot, hormonal analysis and whole-genome sequencing were done in order to elucidate the effects of mtHtt. Results: Evidence for fertility failure of both TgHD generations was observed at the age of 13 months. Reproductive parameters declined and progressively worsened with age. EM revealed numerous pathological features in sperm tails and in testicular epithelium from 24- and 36-month-old TgHD boars. Moreover, immunohistochemistry confirmed significantly lower proliferation activity of spermatogonia in transgenic testes. mtHtt was highly expressed in spermatozoa and testes of TgHD boars and localized in all cells of seminiferous tubules. Levels of fertility-related hormones did not differ in TgHD and WT siblings. Genome analysis confirmed that insertion of the lentiviral construct did not interrupt any coding sequence in the pig genome. Conclusions: The sperm and testicular degeneration of TgHD boars is caused by gain-of-function of the highly expressed mtHtt.

Revelation of the IFNα, IL-10, IL-8 and IL-1β as promising biomarkers reflecting immuno-pathological mechanisms in porcine Huntington's disease model

Studies on Huntington's disease (HD) demonstrated altered immune response in HD gene carriers. Using multiplexing immunoassay, we simultaneously investigated seven cytokines in secretomes of microglia and blood monocytes, cerebrospinal fluid (CSF) and serum collected from transgenic HD minipigs at pre-symptomatic disease stage. Decline in IFNα and IL-10 was observed in CSF and secretome of microglia whilst elevated IL-8 and IL-1β levels were secreted by microglia. Additionally, IL-8 was increased in serum. The proportion of mutant huntingtin in microglia may have causative impact on cytokine production. IFNα, IL-10, IL-8 and IL-1β represent promising biomarkers reflecting immuno-pathological mechanisms in porcine HD model.

Minipig model of Huntington's disease: ¹H magnetic resonance spectroscopy of the brain.

Huntington's disease (HD) is an inherited autosomal neurodegenerative disorder affecting predominantly the brain, characterized by motor dysfunctions, behavioral and cognitive disturbances. The aim of this study was to determine changes in the brain of transgenic minipigs before HD onset using (1)H magnetic resonance (MR) spectroscopy. Measurements were performed on a 3 T MR scanner using a single voxel spectroscopy sequence for spectra acquisition in the white matter and chemical shift imaging sequence for measurement in the striatum, hippocampus and thalamus. A decrease of (phospho)creatine (tCr) concentration was found only in the thalamus (p=0.002) of transgenic minipigs, nevertheless we found significant changes in metabolite ratios. Increase of the ratio choline compounds (tCho)/tCr was found in all examined areas: striatum (p=0.010), thalamus (p=0.011) as well as hippocampus (p=0.027). The ratio N-acetylaspartate+N-acetylaspartylglutamate (tNAA)/tCr (p=0.043) and glutamate+glutamine (Glx)/tCr (p=0.039) was elevated in the thalamus, the ratio myo-inositol (Ins)/tCr (p=0.048) was significantly increased in the hippocampus. No significant differences were observed in the metabolite concentrations in the white matter, however we found significant increase of ratios tNAA/tCr (p=0.018) and tCho/tCr (p=0.003) ratios in transgenic boars. We suppose that the majority of the observed changes are predominantly related to changes in energy metabolism caused by decrease of tCr.

Behavioral phenotyping of minipigs transgenic for the Huntington gene

BackgroundWhile several novel therapeutic approaches for HD are in development, resources to conduct clinical trials are limited. Large animal models have been proposed to improve assessment of safety, tolerability and especially to increase translational reliability of efficacy signals obtained in preclinical studies. They may thus help to select candidates for translation to human studies. We here introduce a battery of novel tests designed to assess the motor, cognitive and behavioral phenotype of a transgenic (tg) HD minipig model. New methodsA group of tgHD and wildtype (wt) Libechov minipigs (n=36) was available for assessment with (1) a gait test using the GAITRite® automated acquisition system, (2) a hurdle-test, (3) a tongue coordination test, (4) a color discrimination test, (5) a startbox back and forth test and (6) a dominance test. Performance of all tests and definition of measures obtained is presented. ResultsMinipigs were able to learn performance of all tests. All tests were safe, well tolerated and feasible. Exploratory between group comparisons showed no differences between groups of tgHD and wt minipigs assessed, but low variability within and between groups. Comparison with existing method(s)So far there are no established or validated assessments to test minipigs in the domains described. ConclusionsThe data shows that the tests presented are safe, well tolerated and all measures defined can be assessed. Prospective longitudinal application of these tests is warranted to determine their test–retest reliability, sensitivity and validity in assessing motor, cognitive and behavioral features of tg and wt minipigs.

Different Forms of Huntingtin in the Most Affected Organs; Brain and Testes of Transgenic Minipigs

Different Forms of Huntingtin in the Most Affected Organs; Brain and Testes of Transgenic Minipigs

Transgenic Wuzhishan minipigs designed to express a dominant-negative porcine growth hormone receptor display small stature and a perturbed insulin/IGF-1 pathway.

Growth hormone (GH) is an anabolic mitogen with widespread influence on cellular growth and differentiation as well as on glucose and lipid metabolism. GH binding to the growth hormone receptor (GHR) on hepatocytes prompts expression of insulin growth factor I (IGF-1) involved in nutritionally induced compensatory hyperplasia of pancreatic β-cell islets and insulin release. A prolonged hyperactivity of the IGF-1/insulin axis in the face of insulinotropic nutrition, on the other hand, can lead to collapse of the pancreatic islets and glucose intolerance. Individuals with Laron syndrome carry mutations in the GHR gene resulting in severe congenital IGF-1 deficiency and elevated GH serum levels leading to short stature as well as perturbed lipid and glucose metabolism. However, these individuals enjoy a reduced prevalence of acne, cancer and possibly diabetes. Minipigs have become important biomedical models for human conditions due to similarities in organ anatomy, physiology, and metabolism relative to humans. The purpose of this study was to generate transgenic Wuzhishan minipigs by handmade cloning with impaired systemic GHR activity and assess their growth profile and glucose metabolism. Transgenic minipigs featuring overexpression of a dominant-negative porcine GHR (GHR(dm)) presented postnatal growth retardation and proportionate dwarfism. Molecular changes included elevated GH serum levels and mild hyperglycemia. We believe that this model may prove valuable in the study of GH functions in relation to cancer, diabetes and longevity.

Postmortem findings in cloned and transgenic piglets dead before weaning

Important factors contributing to the well-known high mortality of piglets produced by SCNT are gross malformations of vital organs. The aim of the present retrospective study was to describe malformations found in cloned piglets, transgenic or not, dying or culled before weaning on Day 28. Large White (LW) embryos were transferred to 78 LW recipients, while 72 recipients received Göttingen embryos (67 transgenic and five not transgenic) and 56 received Yucatan embryos (43 transgenic and 13 not transgenic). Overall pregnancy rate was 76%, and there were more abortions in recipients with minipig embryos than in those with LW embryos (26% and 24% vs. 6%). Piglets (n = 815) were born from 128 sows with 6.5 ± 0.4 full-born piglets per litter. The overall rate of stillborn piglets was 21% of all born with the number of stillborn piglets ranging from one to nine in a litter. The mortality of the surviving piglets during the first month was 48%. Thus, altogether 58% of the full-born piglets died before weaning. In 87 of the 128 litters (68%), one to 12 of the piglets showed major or minor malformations. Malformations were found in 232 piglets (29.5% of all born). A single malformation was registered in 152 piglets, but several piglets showed two (n = 58) or more (n = 23) malformations (7.4% and 2.8% of all born, respectively). A significantly higher malformation rate was found in transgenic Göttingen and Yucatan piglets (32% and 46% of all born, respectively) than in nontransgenic LW (17%). There was a gender difference in the transgenic minipigs because male piglets had a higher rate of malformations (49.1%) than females (29.7%). The most common defects in the cloned piglets were in the digestive (12.2%), circulatory (9.4%), reproductive (11.3%), and musculoskeletal (9.1%) systems. Malformations of the musculoskeletal system were most frequent in Göttingen (16.3% vs. approximately 5.5% in the two other breeds), whereas abnormal cardiopulmonary systems were most frequent in Yucatan piglets (26.9% vs. 2.1% in LW and 5.3% in Göttingen). In conclusion, these results show that pig cloning results in a considerable loss of piglets and that many of these can be related to various malformations that all are also seen in noncloned piglets. Because approximately half of the cloned piglets still survive, even with eventual unknown minor malformations, use of pigs as models for human diseases is still realistic. However, continued efforts are needed to further reduce the level of malformations.

Production of hGFAP-DsRed transgenic Guangxi Bama mini-pigs via somatic cell nuclear transfer.

The mini-pig is a useful animal model for human biomedical research due to its physiological similarity to humans and the ease of handling. In order to optimize the efficiency of production of transgenic Bama mini-pigs through somatic cell nuclear transfer (SCNT), we examined the effects of contact inhibition, roscovitine treatment, and serum starvation on the cell cycle synchronization and transgenic cloned embryo development in vivo and in vitro after nuclear transfer. The analysis showed that the rates of G0/G1 stage cells in the contact inhibition (92.11%) and roscovitine treatment groups (89.59%) were significantly higher than in serum starvation group (80.82%). A higher rate of apoptosis was seen in the serum starvation group (14.13%) compared to the contact inhibition and roscovitine treatment groups (6.71 and 2.46% respectively, P < 0.05). There was a significant decrease in blastocyst yield in the serum starvation group (14.19%) compared to the roscovitine treatment and contact inhibition groups (21.31 and 20.32% respectively, P < 0.05). A total of 1070 transgenic cloned embryos derived from the three treatment groups were transferred to surrogate sows; one pregnancy was established and three embryos from the roscovitine treatment group successfully completed gestation. These results indicate that the roscovitine treatment was more effective at synchronizing transgenic kidney cells in Bama mini-pigs and allowed reconstructed embryos to develop to full term.

Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig.

ABSTRACTAnimal models of familial juvenile onset of Alzheimer's disease (AD) often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw). We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs) isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs) from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

Abstract 17809: In Vivo High Resolution Isotropic 3D MRI of Coronary Atherosclerosis in Hypertensive Hypercholesterolemic Minipigs

Introduction: Accurate non-invasive visualization and quantification of coronary atherosclerosis may greatly facilitate clinical research and practice, but the lack of suitable human-sized atherosclerosis models with progressive coronary lesions has hampered development and validation of reliable imaging techniques. Methods: D374Y-PCSK9 transgenic minipigs with (n=7) or without (n=8) hypertension, induced by suprarenal aortic banding, were placed on a cholesterol-rich diet for 16 months. Wildtype minipigs (n=4) on normal diet served as disease-free controls. A T1-weighted inversion recovery fast gradient echo magnetic resonance imaging (MRI) sequence was used to acquire 1x1x1mm images of the coronary arteries. MRI scans were conducted following injection of a gadolinium labeled elastin specific contrast agent (LMI1174). Pigs were subsequently euthanized and the coronary arteries were processed for pathological analysis. LMI1174 was provided by Lantheus Medical Imaging under a material transfer agreement Results: Hypertension significantly augmented coronary fibroatheromas by more than 5-fold compared to normotensive transgenic pigs. No signal enhancement was observed in lesion-free coronaries of wildtype pigs. In hypertensive pigs, there was a strong correlation between plaque area measured by MRI and plaque area measured postmortem r2=0.84, p=0.02 (Fig 1). Conclusions: Hypertensive transgenic minipigs with accelerated coronary atherosclerosis provide a useful model for coronary imaging. Using this model we were able to develop a high-resolution isotropic LMI1174-enhanced scanning technique that accurately depicts coronary atherosclerotic lesion size and distribution. Figure 1: A) Digitally unfolded and color coded MRI of the LAD (left) and corresponding sudan IV stained segment opened and viewed en face (right). B) Cross-sectional MRI of another LAD and corresponding histological sections showing fibroatheromas.

C11 Transgenic Minipigs For N-terminal Part Of Human Mutated Huntingtin: Following Disease Development

Background In order to facilitate studies of pathogenesis and therapy of Huntington’s disease (HD), we have generated transgenic minipigs using microinjection of a lentiviral vector encoding N-terminal (548 aa) part of human huntingtin (Htt) containing 126 CAG/CAA repeats under the control of the human Htt promoter. The progression of HD in human patients is predominantly linked with formation of aggregates, oligomers, and fragments of Htt. Among other factors corresponding to HD is lowered BDNF (brain-derived neurotrophic factor) expression in brain, accumulation of ACBD3 (acyl-CoA binding domain containing 3), and formation of toxic Rhes/mtHtt/ACBD3 complex. Aim To follow disease development in transgenic (TgHD) minipigs using specific biomarkers of HD. Methods At present, TgHD and WT (wild-type) minipigs of F0, F1, F2 and F3 generations are being used for invasive (24 and 36 months old animals) and non-invasive approaches (up to five years old animals). We perform biochemical assays on tissues (with main focus on brain) and cells (fibroblasts, and buccal swabs) in order to follow HD development in our model. Results To date, we have not been successful in the aggregate detection, which could be due to the young age of TgHD animals. However, we have detected Htt oligomers, several Htt fragments of lower molecular weight in samples from TgHD minipigs and not from WT siblings. In addition, we monitor altered levels of proteins previously reported as markers of HD development, namely BDNF and Rhes/mtHtt/ACBD3 complex Conclusion We provide complex information about HD phenotype development in transgenic minipig model between 24–60 months of age. Support CHDI Foundation (A-5378), TACR (TA01011466), European Regional Development FundCZ.1.05/2.1.00/03.0124

C12 Volumetry of Nucleus Caudatus, Lateral Ventricles and Cerebrum of Founder and Second Generation Libechov Transgenic HD Minipigs

<h3>Background</h3> Transgenic HD minipigs were examined with Magnetic Resonance Imaging for phenotyping the disease model. MRI scans were performed on the founder and second generation of transgenic HD minipigs from the Institute of Animal Physiology and Genetics in Libéchov (Czech Republic). These scans include anatomical, diffusion-weighted and spectroscopic sequences. <h3>Aim</h3> For phenotyping, different brain regions of the transgenic HD and wildtype minipigs were investigated periodically. <h3>Methods/techniques</h3> Three transgenic HD and two wildtype minipigs were scanned on a 3T Philips Achieva system three times within four years. A 3D gradient echo data set was acquired in 32 min scan time using the following parameters: slices: 450, reconstructed voxel size: 0.5 mm isotropic, FOV: 296 × 296 × 225, NSA: 3, TR: 7.5 ms, TE: 3.4 ms, flip angle: 9 deg. The nucleus caudatus, the lateral ventricles and the cerebrum were segmented semi-manually using the software package “Amira®” and the corresponding volumes were calculated. <h3>Results/outcome</h3> So far no significant differences between the transgenic (n = 3) and wildtype (n = 2) group in the analysed volumes were observed. <h3>Conclusions/discussion</h3> In this pilot study, no changes in the brain regions assessed were identified. However, the very small number of animals included limits these observations. Due to the slowly progressive nature of HD, changes in the volume between transgenic and wildtype minipigs may be expected to be expressed at later time points. <h3>Acknowledgement</h3> Funded by the CHDI Foundation.

C15 Screening Of Huntington's Disease Markers In The Brain Of Transgenic Minipigs For The Human Mutated Huntingtin (f2 Generation, Age 24 Months)

<h3>Background</h3> The discovery of the huntingtin gene enabled the creation of transgenic HD models including the transgenic minipigs (TgHD) used in this study. The advantages of minipigs as models of HD disease are that their brains are very similar to humans in terms of anatomy, histology, lifespan and genetics. The brain, especially its parts such as striatum and cortex, are considered to be mostly affected by HD. <h3>Aim</h3> To investigate the expression of selected HD markers: mutated huntingtin (mtHtt) and their aggregates, Golgi resistant protein GCP60 and microgliosis in the brain (cortex and striatum) of transgenic minipigs for the human mtHtt and compare them with brains of wild type (WT) minipigs. <h3>Methods</h3> In this study we used WT and TgHD minipigs for N-terminal part of the human mtHtt (548aaHTT-145Q, both F2 generation, age 24 months). HD markers were examined immunohistochemically (IHC) on cryostat sections or biochemically by western blotting (WB) using the following primary antibodies: anti-polyglutamines, anti-polyglutamine-expansion diseases marker, aggregated huntingtin (S829), anti-ACBD3 (GPC60), anti-IBA1. <h3>Results</h3> The IHC examination showed increased expression of polyglutamines, GCP60 and IBA1 proteins in the brain tissue of TgHD minipigs. The WB examination confirmed massive polyglutamine expression and increased accumulation of Golgi resistant protein GCP60 in the TgHD brain tissue. Also we revealed higher number of activated microglia in nucleus caudate of TgHD in comparison with WT minipigs. No mtHtt aggregates were detected in TgHD brains. <h3>Conclusion</h3> Using IHC and WB methods we were able to characterise early changes in the brain tissue of TgHD animals of F2 generation. This animal study of initial stages of HD development at the cellular and molecular level can contribute to better understanding of human disease pathology. <h3>Support</h3> CHDI Foundation (A-5378), TACR (TA01011466), European Regional Development FundCZ.1.05/2.1.00/03.0124, RVO 67985904.

Generation of AQP2-Cre transgenic mini-pigs specifically expressing Cre recombinase in kidney collecting duct cells

The important differences in physiological parameters and anatomical characteristics of the kidney between humans and mice make it difficult to replicate the precise progression of human renal cystic diseases in gene modification mouse models. In contrast to mice, pigs are a better animal model of human diseases, as they are more similar in terms of organ size, structure, and physiological parameters. Here, we report the generation and initial examination of an AQP2-Cre transgenic (Tg) Chinese miniature (mini)-pig line that expresses Cre recombinase exclusively in kidney collecting duct cells. An 8-kb fragment of the mini-pig aquaporin 2 (AQP2) 5'-flanking region was utilized to direct Cre expression in Tg mini-pigs. Two Tg mini-pigs were generated by pig somatic cell nuclear transfer and both carried the entire coding sequence of Cre recombinase. RT-PCR and western blotting analysis revealed that Cre recombinase was uniquely expressed in the kidney, while immunohistochemical studies located its expression in kidney collecting duct cells. Furthermore, six integration sites and 12-14 copies of the Cre gene were detected in various tissues by high-efficiency thermal asymmetric interlaced PCR and absolute quantitative real-time PCR, respectively. Combined with previous studies of Cre recombinase activity, we believe that this AQP2-Cre Tg mini-pig line will be a useful tool to generate kidney collecting duct cell-specific gene knockout mini-pig models, thereby allowing the investigation of gene functions in kidney development and the mechanisms of human renal cystic disease.

Development of Transgenic Minipigs with Expression of Antimorphic Human Cryptochrome 1

Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3–20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1AP). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders.

Expression of Huntingtons disease markers in the cornea of minipig transgenic for the human mutated huntingtin

AbstractPurpose Huntingtons disease (HD) is a neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) domain in the protein of huntingtin (htt). PolyQ expansion above 35–40 results in disease associated with htt aggregation into inclusion bodies. The recent studies have shown that the illness arises not only in neurons, but also in non‐neuronal cells. Purpose of this study was to investigate the expression of selected HD markers (on htt and their aggregates, ferritin and ubiquitin) in corneas of minipig transgenic for the human mutated huntingtin and compare them with corneas of wild type (WT) minipig.Methods In this study we used WT minipigs and minipigs transgenic for N‐terminal part of the human mutated huntingtin (both F2 generation, age 24 months). HD markers were examined on cryostat sections immunohistochemically by using the following primary antibodies: anti‐polyglutamines, anti‐polyglutamine‐expansion diseases marker, MW8 marker, anti‐ferritin and anti‐ubiguitin.Results Immunohistochemical examination on huntingtin demonstrated the increased expression of this protein in corneal epithelium and keratocytes of transgenic minipig in comparison with wild type cornea. The expression of ferritin detected in epithelial cells was very low in both studied corneas without significant difference between them. No nuclear inclusions or ubiquitous proteins were found in the cornea of transgenic minipig.Conclusion This study showed that preclinical (silent) stages of HD are characterized by an elevated level of mutagenic huntingtin without formation of nuclear inclusions and ubiquitination of proteins. Supported by CHDI Foundation (A‐5378 and A‐5379), TA01011466, CZ.1.05./2.1.00/03.0124 and RVO: 67985904.

Improvement of Cloning Efficiency in Minipigs Using Post-thawed Donor Cells Treated with Roscovitine

Massachusetts General Hospital miniature pigs (MGH minipigs) have been established for organ transplantation studies across the homozygous major histocompatibility complex, but cloning efficiency of MGH minipigs is extremely low. This study was designed to increase the productivity of MGH minipigs by nuclear transfer of post-thaw donor cells after 1h co-incubation with roscovitine. The MGH minipig cells were genetically modified with GT KO (alpha1,3-galactosyltransferase knock-out) and hCD46 KI (human CD46 knock-in) and used as donor cells. The GT KO/hCD46 KI donor cells were cultured for either 3days (control group) or 1h after thawing with 15μM roscovitine (experimental group) prior to the nuclear transfer. The relative percentage of the transgenic donor cells that entered into G0/G1 was 93.7% (±2.54). This was different from the donor cells cultured for 1h with the roscovitine-treated group (84.6% ±4.6) (P<0.05) and without roscovitine (78.6% ±5.5) (P<0.01), respectively. The pregnancy rate and delivery rate in the roscovitine group (8/12 and 6/8, respectively) were significantly higher (P<0.01) than those in the control group (6/19 and 3/6, respectively). In the experimental group, 12 GT KO/hCD46 KI transgenic minipigs were successfully generated, and five minipigs among them survived for more than 6months so far. The recipient-based individual cloning efficiency ranged from 0.74 to 2.54%. In conclusion, gene-modified donor cells can be used for cloning of MGH minipigs if the cells are post-thawed and treated with roscovitine for 1h prior to nuclear transfer.