PURPOSE: This study aimed to evaluate early-phase safety of subretinal application of AAVanc80.CAG.USH1Ca1 (OT_USH_101) in wild-type (WT) pigs, examining the effects of a vehicle control, low dose, and high dose. METHODS: Twelve WT pigs (24 eyes) were divided into three groups: four pigs each received bilateral subretinal injections of either vehicle, low dose (3.3 × 1010 vector genomes [vg] per eye), or high dose (1.0 × 1011 vg per eye). Total retinal thickness (TRT) was evaluated using optical coherence tomography and retinal function was assessed with full-field electroretinography (ff-ERG) at baseline and two months post-surgery. After necropsy, retinal changes were examined through histopathology, and human USH1C_a1/harmonin expression was assessed by quantitative PCR (qPCR) and Western blotting. RESULTS: OT_USH_101 led to high USH1C_a1 expression in WT pig retinas without significant TRT changes two months after subretinal injection. The qPCR revealed expression of the human USH1C_a1 transgene delivered by the adeno-associated virus vector. TRT changes were minimal across groups: vehicle (256 ± 21 to 243 ± 18 μm; P = 0.108), low dose (251 ± 32 to 258 ± 30 μm; P = 0.076), and high dose (242 ± 24 to 259 ± 28 μm; P = 0.590). The ff-ERG showed no significant changes in rod or cone responses. Histopathology indicated no severe retinal adverse effects in the vehicle and low dose groups. CONCLUSIONS: Early-phase clinical imaging, electrophysiology, and histopathological assessments indicated that subretinal administration of OT_USH_101 was well tolerated in the low-dose treatment arm. OT_USH_101 treatment resulted in high expression of human USH1C_a1. Although histopathological changes were not severe, more frequent changes were observed in the high-dose group.

• MeSH
• Cytoskeletal Proteins genetics   MeSH
• Dependovirus genetics   MeSH
• Electroretinography * MeSH
• Genetic Therapy methods   MeSH
• Genetic Vectors * MeSH
• Injections, Intraocular * MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Tomography, Optical Coherence * MeSH
• Swine MeSH
• Cell Cycle Proteins genetics   MeSH
• Gene Expression Regulation MeSH
• Retina * metabolism   pathology   MeSH
• Transgenes * MeSH
• Blotting, Western MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

• MeSH
• Actins genetics   metabolism   MeSH
• Cytomegalovirus Infections * genetics   metabolism   MeSH
• Dependovirus genetics   metabolism   MeSH
• Genetic Vectors genetics   MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Parvovirinae * genetics   MeSH
• Retinal Ganglion Cells metabolism   MeSH
• Transduction, Genetic MeSH
• Transgenes MeSH
• Green Fluorescent Proteins genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Adeno-associated viral vectors are widely used as vehicles for gene transfer to the nervous system. The promoter and viral vector serotype are two key factors that determine the expression dynamics of the transgene. A previous comparative study has demonstrated that AAV1 displays efficient transduction of layer V corticospinal neurons, but the optimal promoter for transgene expression in corticospinal neurons has not been determined yet. In this paper, we report a side-by-side comparison between four commonly used promoters: the short CMV early enhancer/chicken β actin (sCAG), human cytomegalovirus (hCMV), mouse phosphoglycerate kinase (mPGK) and human synapsin (hSYN) promoter. Reporter constructs with each of these promoters were packaged in AAV1, and were injected in the sensorimotor cortex of rats and mice in order to transduce the corticospinal tract. Transgene expression levels and the cellular transduction profile were examined after 6 weeks. The AAV1 vectors harbouring the hCMV and sCAG promoters resulted in transgene expression in neurons, astrocytes and oligodendrocytes. The mPGK and hSYN promoters directed the strongest transgene expression. The mPGK promoter did drive expression in cortical neurons and oligodendrocytes, while transduction with AAV harbouring the hSYN promoter resulted in neuron-specific expression, including perineuronal net expressing interneurons and layer V corticospinal neurons. This promoter comparison study contributes to improve transgene delivery into the brain and spinal cord. The optimized transduction of the corticospinal tract will be beneficial for spinal cord injury research.

• MeSH
• Dependovirus * genetics   MeSH
• Genetic Vectors genetics   MeSH
• Rats MeSH
• Mice MeSH
• Promoter Regions, Genetic MeSH
• Pyramidal Tracts * MeSH
• Transduction, Genetic MeSH
• Transgenes MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.

• MeSH
• Biotechnology methods   MeSH
• Genetic Engineering methods   MeSH
• Disease Resistance * MeSH
• Prunus genetics   virology   MeSH
• Transgenes MeSH
• Gene Silencing * MeSH
• Capsid Proteins genetics   metabolism   MeSH
• Plum Pox Virus genetics   pathogenicity   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Fatty acid desaturase 2 (Fads2) is the key enzyme of long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis. Endogenous production of these biomolecules in vertebrates, if present, is insufficient to meet demand. Hence, LC-PUFA are considered as conditionally essential. At present, however, LC-PUFA are globally limited nutrients due to anthropogenic factors. Research attention has therefore been paid to finding ways to maximize endogenous LC-PUFA production, especially in production species, whereby deeper knowledge on molecular mechanisms of enzymatic steps involved is being generated. This review first briefly informs about the milestones in the history of LC-PUFA essentiality exploration before it focuses on the main aim-to highlight the fascinating Fads2 potential to play roles fundamental to adaptation to novel environmental conditions. Investigations are summarized to elucidate on the evolutionary history of fish Fads2, providing an explanation for the remarkable plasticity of this enzyme in fish. Furthermore, structural implications of Fads2 substrate specificity are discussed and some relevant studies performed on organisms other than fish are mentioned in cases when such studies have to date not been conducted on fish models. The importance of Fads2 in the context of growing aquaculture demand and dwindling LC-PUFA supply is depicted and a few remedies in the form of genetic engineering to improve endogenous production of these biomolecules are outlined.

• MeSH
• Fatty Acid Desaturases chemistry   metabolism   MeSH
• Phylogeny MeSH
• Genetic Engineering MeSH
• Fatty Acids, Unsaturated chemistry   MeSH
• Gene Expression Regulation MeSH
• Fish Proteins chemistry   metabolism   MeSH
• Fishes * MeSH
• Substrate Specificity MeSH
• Transgenes MeSH
• Aquaculture MeSH
• Structure-Activity Relationship MeSH
• Inflammation MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Long transgenes are often used in mammalian genetics, e.g., to rescue mutations in large genes. In the course of experiments addressing the genetic basis of hybrid sterility caused by meiotic defects in mice bearing different alleles of Prdm9, we discovered that introduction of copy-number variation (CNV) via two independent insertions of long transgenes containing incomplete Prdm9 decreased testicular weight and epididymal sperm count. Transgenic animals displayed increased occurrence of seminiferous tubules with apoptotic cells at 18 days postpartum (dpp) corresponding to late meiotic prophase I, but not at 21 dpp. We hypothesized that long transgene insertions could cause asynapsis, but the immunocytochemical data revealed that the adult transgenic testes carried a similar percentage of asynaptic pachytene spermatocytes as the controls. These transgenic spermatocytes displayed less crossovers but similar numbers of unrepaired meiotic breaks. Despite slightly increased frequency of metaphase I spermatocytes with univalent chromosome(s) and reduced numbers of metaphase II spermatocytes, cytological studies did not reveal increased apoptosis in tubules containing the metaphase spermatocytes, but found an increased percentage of tubules carrying apoptotic spermatids. Sperm counts of subfertile animals inversely correlated with the transcription levels of the Psmb1 gene encoded within these two transgenes. The effect of the transgenes was dependent on sex and genetic background. Our results imply that the fertility of transgenic hybrid animals is not compromised by the impaired meiotic synapsis of homologous chromosomes, but can be negatively influenced by the increased expression of the introduced genes.

• MeSH
• Apoptosis genetics   MeSH
• DNA Breaks, Double-Stranded MeSH
• Fertility genetics   MeSH
• Genetic Background MeSH
• Cell Cycle Checkpoints genetics   MeSH
• Mice MeSH
• Pachytene Stage genetics   MeSH
• Sperm Count MeSH
• Spermatocytes metabolism   MeSH
• Testis anatomy & histology   metabolism   MeSH
• Transgenes * MeSH
• DNA Copy Number Variations * MeSH
• Organ Size MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The natural behavior of mesenchymal stem cells (MSCs) and their exosomes in targeting tumors is a promising approach for curative therapy. Human tumor tropic mesenchymal stem cells (MSCs) isolated from various tissues and MSCs engineered to express the yeast cytosine deaminase::uracil phosphoribosyl transferase suicide fusion gene (yCD::UPRT-MSCs) released exosomes in conditional medium (CM). Exosomes from all tissue specific yCD::UPRT-MSCs contained mRNA of the suicide gene in the exosome's cargo. When the CM was applied to tumor cells, the exosomes were internalized by recipient tumor cells and in the presence of the prodrug 5-fluorocytosine (5-FC) effectively triggered dose-dependent tumor cell death by endocytosed exosomes via an intracellular conversion of the prodrug 5-FC to 5-fluorouracil. Exosomes were found to be responsible for the tumor inhibitory activity. The presence of microRNAs in exosomes produced from naive MSCs and from suicide gene transduced MSCs did not differ significantly. MicroRNAs from yCD::UPRT-MSCs were not associated with therapeutic effect. MSC suicide gene exosomes represent a new class of tumor cell targeting drug acting intracellular with curative potential.

• MeSH
• Cytosine Deaminase genetics   metabolism   MeSH
• Exosomes genetics   metabolism   MeSH
• Flucytosine metabolism   MeSH
• Fluorouracil metabolism   pharmacology   MeSH
• Fungal Proteins genetics   metabolism   MeSH
• Genetic Therapy methods   MeSH
• Yeasts genetics   metabolism   MeSH
• Humans MeSH
• Mesenchymal Stem Cells metabolism   MeSH
• Cell Line, Tumor MeSH
• Breast Neoplasms genetics   metabolism   pathology   MeSH
• Pentosyltransferases genetics   metabolism   MeSH
• Prodrugs metabolism   MeSH
• Cell Proliferation drug effects   genetics   MeSH
• Antimetabolites, Antineoplastic metabolism   pharmacology   MeSH
• Genes, Transgenic, Suicide genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3-GFP, pOBCol3.6-GFP, and DMP1-GFP), similar to the endogenous proteins, are also expressed by bone-forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP-Cerulean/DMP1-Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24-258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1-Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP-Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts.

• MeSH
• Cell Differentiation MeSH
• Extracellular Matrix Proteins genetics   MeSH
• Fluorescent Dyes MeSH
• Phosphoproteins genetics   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Odontoblasts cytology   MeSH
• Genes, Reporter * MeSH
• Sialoglycoproteins genetics   MeSH
• Transgenes MeSH
• Green Fluorescent Proteins genetics   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, N.I.H., Extramural MeSH

Use of chemically inducible systems for transgene expression is a crucial requirement for modern plant biology research, as it allows (1) expression of transgenes that compromise plant viability or fertility when constitutively expressed and (2) spatiotemporal control of transgene expression levels. We describe the stringently regulated and highly responsive dexamethasone-inducible gene expression system pOp6/LhGR, which comprises the chimeric transcription activator LhGR and the corresponding pOp6 promoter. Upon induction, the LhGR activator binds to the pOp6 promoter and induces expression of the target gene of interest. We provide detailed protocols for inducing transgene expression at different developmental stages and in different plant species and discuss dexamethasone stability and use of its analogs. We also introduce new, versatile, GATEWAY-compatible binary vectors that are now available for the pOp6/LhGR system. © 2019 by John Wiley & Sons, Inc.

• MeSH
• Transcriptional Activation * drug effects   MeSH
• Arabidopsis genetics   MeSH
• Dexamethasone pharmacology   MeSH
• DNA-Binding Proteins genetics   MeSH
• Genetic Techniques MeSH
• Plants, Genetically Modified genetics   MeSH
• Lac Repressors genetics   MeSH
• Promoter Regions, Genetic MeSH
• Escherichia coli Proteins genetics   MeSH
• Receptors, Glucocorticoid genetics   MeSH
• Saccharomyces cerevisiae Proteins genetics   MeSH
• Transgenes * MeSH
• Transcription Factors genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Hypertension, dyslipidemia, and insulin resistance in the spontaneously hypertensive rat (SHR) can be alleviated by rescuing CD36 fatty acid translocase. The present study investigated whether transgenic rescue of CD36 in SHR could affect mitochondrial function and activity of selected metabolic enzymes in the heart. These analyses were conducted on ventricular preparations derived from SHR and from transgenic strain SHR-Cd36 that expresses a functional wild-type CD36. Our respirometric measurements revealed that mitochondria isolated from the left ventricles exhibited two times higher respiratory activity than those isolated from the right ventricles. Whereas, we did not observe any significant changes in functioning of the mitochondrial respiratory system between both rat strains, enzyme activities of total hexokinase, and both mitochondrial and total malate dehydrogenase were markedly decreased in the left ventricles of transgenic rats, compared to SHR. We also detected downregulated expression of the succinate dehydrogenase subunit SdhB (complex II) and 70 kDa peroxisomal membrane protein in the left ventricles of SHR-Cd36. These data indicate that CD36 may affect in a unique fashion metabolic substrate flexibility of the left and right ventricles.

• MeSH
• ATP-Binding Cassette Transporters genetics   metabolism   MeSH
• CD36 Antigens genetics   metabolism   MeSH
• Gene Expression MeSH
• Hexokinase genetics   metabolism   MeSH
• Hypertension enzymology   genetics   physiopathology   MeSH
• Insulin Resistance MeSH
• Myocytes, Cardiac enzymology   pathology   MeSH
• Rats MeSH
• Malate Dehydrogenase genetics   metabolism   MeSH
• Mitochondria enzymology   pathology   MeSH
• Oxidative Phosphorylation MeSH
• Rats, Inbred SHR MeSH
• Rats, Transgenic MeSH
• Primary Cell Culture MeSH
• Gene Expression Regulation MeSH
• Oxygen Consumption genetics   MeSH
• Heart Ventricles enzymology   pathology   MeSH
• Succinate Dehydrogenase genetics   metabolism   MeSH
• Transgenes * MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH