OBJECTIVE: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. METHODS: The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. RESULTS: rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. CONCLUSION Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.
Mice ; Animals ; Humans ; Melitten/genetics* ; Dependovirus/genetics* ; Serogroup ; HEK293 Cells ; Mice, Nude ; Mice, Inbred C57BL ; Transgenes ; Genetic Vectors/genetics*

With the development of various biotechnology,the research on molecular genetics of medicinal plants has gradually deepened. In this paper,the research system of molecular genetics of medicinal plants was proposed for the first time,which was elaborated from the aspects of genetic resources,genome,gene function and research methods. The application fields of medicinal plant mainly contain species identification,molecular breeding and biosynthesis. The research directions of molecular genetics of medicinal plants in genetic resources,model platform,synthetic biology and molecular breeding were put forward,which include 1 000 genome projects of medicinal plants,model species and mutant libraries,gene original libraries of heterologous synthetic systems,construction gene original library and specific chassis cells in heterologous synthesis system of active ingredient,breeding of new varieties of medicinal plants with high active ingredient and high resistance based on molecular markers andtransgenes.
Biotechnology ; Gene Library ; Genetic Markers ; Genome, Plant ; Molecular Biology ; trends ; Plant Breeding ; Plants, Medicinal ; genetics ; Research ; Transgenes

BACKGROUND: Osteoarthritis (OA) is a highly prevalent degenerative joint disease involving joint cartilage and its surrounding tissues. OA is the leading cause of pain and disability worldwide. At present, there are no disease-modifying OA drugs, and the primary therapies include exercise and nonsteroidal anti-inflammatory drugs until total joint replacement at the end-stage of the disease. METHODS: In this review, we summarized the current state of knowledge in genetic and epigenetic associations and risk factors for OA and their potential diagnostic and therapeutic applications. RESULTS: Genome-wide association studies and analysis of epigenetic modifications (such as miRNA expression, DNA methylation and histone modifications) conducted across various populations support the notion that there is a genetic basis for certain subsets of OA pathogenesis. CONCLUSION: With recent advances in the development of genome editing technologies such as the CRISPR-Cas9 system, these genetic and epigenetic alternations in OA can be used as platforms from which potential biomarkers for the diagnosis, prognosis, drug response, and development of potential personalized therapeutic targets for OA can be approached. Furthermore, genome editing has allowed the development of “designer” cells, whereby the receptors, gene regulatory networks, or transgenes can be modified as a basis for new cell-based therapies.
Biomarkers ; Cartilage ; Diagnosis ; DNA Methylation ; Epigenomics ; Gene Regulatory Networks ; Genetics ; Genome ; Genome-Wide Association Study ; Histones ; Humans ; Joint Diseases ; Joints ; MicroRNAs ; Osteoarthritis ; Precision Medicine ; Prognosis ; Risk Factors ; Transgenes

We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.
Arabidopsis/genetics* ; Arabidopsis Proteins/metabolism* ; Cloning, Molecular ; DNA, Complementary/metabolism* ; Flowers/physiology* ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Homeodomain Proteins/metabolism* ; Lamiales/physiology* ; MicroRNAs/metabolism* ; Nuclear Proteins/metabolism* ; Plants, Genetically Modified/physiology* ; Plasmids/metabolism* ; Polymerase Chain Reaction ; Transgenes

In plants, lipoxygenases (LOXs) play a crucial role in biotic and abiotic stresses. In our previous study, five 13-LOX genes of oriental melon were regulated by abiotic stress but it is unclear whether the 9-LOX is involved in biotic and abiotic stresses. The promoter analysis revealed that CmLOX09 (type of 9-LOX) has hormone elements, signal substances, and stress elements. We analyzed the expression of CmLOX09 and its downstream genes-CmHPL and CmAOS-in the leaves of four-leaf stage seedlings of the oriental melon cultivar "Yumeiren" under wound, hormone, and signal substances. CmLOX09, CmHPL, and CmAOS were all induced by wounding. CmLOX09 was induced by auxin (indole acetic acid, IAA) and gibberellins (GA₃); however, CmHPL and CmAOS showed differential responses to IAA and GA₃. CmLOX09, CmHPL, and CmAOS were all induced by hydrogen peroxide (H₂O₂) and methyl jasmonate (MeJA), while being inhibited by abscisic acid (ABA) and salicylic acid (SA). CmLOX09, CmHPL, and CmAOS were all induced by the powdery mildew pathogen Podosphaera xanthii. The content of 2-hexynol and 2-hexenal in leaves after MeJA treatment was significantly higher than that in the control. After infection with P. xanthii, the diseased leaves of the oriental melon were divided into four levels-levels 1, 2, 3, and 4. The content of jasmonic acid (JA) in the leaves of levels 1 and 3 was significantly higher than that in the level 0 leaves. In summary, the results suggested that CmLOX09 might play a positive role in the response to MeJA through the hydroperoxide lyase (HPL) pathway to produce C6 alcohols and aldehydes, and in the response to P. xanthii through the allene oxide synthase (AOS) pathway to form JA.
Abscisic Acid ; Acetates/chemistry* ; Aldehyde-Lyases/metabolism* ; Aldehydes/chemistry* ; Cucurbitaceae/genetics* ; Cyclopentanes/chemistry* ; Cytochrome P-450 Enzyme System/metabolism* ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; Hormones/metabolism* ; Hydrogen Peroxide/metabolism* ; Intramolecular Oxidoreductases/metabolism* ; Lipoxygenase/metabolism* ; Oxylipins/chemistry* ; Plant Leaves/genetics* ; Plant Proteins/metabolism* ; Promoter Regions, Genetic ; Salicylic Acid/chemistry* ; Seedlings/metabolism* ; Signal Transduction ; Stress, Physiological ; Transgenes

TRPML1 channel is a non-selective group-2 transient receptor potential (TRP) channel with Ca permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV (MLIV). In the present study, we determined the cryo-electron microscopy (cryo-EM) structures of Mus musculus TRPML1 (mTRPML1) in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain (PMD) may sense the luminal/extracellular stimuli and undergo a "move upward" motion during endocytosis, thus triggering the overall conformational change in TRPML1. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca, and phosphoinositides in a combined manner so as to accommodate the dynamic endocytosis process.
Animals ; Calcium ; metabolism ; Cryoelectron Microscopy ; Endocytosis ; Endosomes ; metabolism ; Gene Expression ; HEK293 Cells ; Humans ; Hydrogen-Ion Concentration ; Lysosomes ; metabolism ; Mice ; Models, Biological ; Mucolipidoses ; genetics ; metabolism ; pathology ; Nanostructures ; chemistry ; ultrastructure ; Phosphatidylinositols ; metabolism ; Transgenes ; Transient Receptor Potential Channels ; chemistry ; genetics ; metabolism

Increasing the production and secretion of endogenous opioid peptide by immune cell can significantly induce myocardial protective effects against ischemia-reperfusion injury. Gene therapy is promising to increase endogenous enkephalin (ENK). However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, gene recombination, oncogene activation, the production of antibacterial antibody and changes in physiological gene expression. Minimalistic immunologically defined gene expression (MIDGE) can overcome all the deficients of viral and plasmid vectors. The exon of rat's preproenkephalin (PPENK) gene was amplified by PCR and the fragments were cloned into pEGFP-N1 plasmids. The recombined plasmids were digested with enzymes to obtain a linear vector contained promoter, preproenkephalin gene, RNA stable sequences and oligodesoxy nucleotides (ODNs) added to both ends of the gene vector to protect gene vector from exonuclease degradation. A nuclear localization sequence (NLS) was attached to an ODN to ensure the effective transport to the nucleus and transgene expression. Flow cytometry, laser confocal microscopy and Western blotting demonstrated that PPENK-MIDGE-NLS can transfect leukocyte of rat in vivo, increase the expression of proenkephalin (PENK) in tissue, and the transfection efficiency depends on gene vector's dosage. These results indicate that PPENK-MIDGE-NLS could be an innovative method to protect and treatment of myocardial ischemia-reperfusion injury.
Animals ; Cloning, Molecular ; Enkephalins ; genetics ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Leukocytes ; Plasmids ; Promoter Regions, Genetic ; Protein Precursors ; genetics ; Rats ; Transfection ; Transgenes

Dentin matrix protein 1 (DMP1) is essential to odontogenesis. Its mutations in human subjects lead to dental problems such as dental deformities, hypomineralization and periodontal impairment. Primarily, DMP1 is considered as an extracellular matrix protein that promotes hydroxyapatite formation and activates intracellular signaling pathway via interacting with αvβ3 integrin. Recent in vitro studies suggested that DMP1 might also act as a transcription factor. In this study, we examined whether full-length DMP1 could function as a transcription factor in the nucleus and regulate odontogenesis in vivo. We first demonstrated that a patient with the DMP1 M1V mutation, which presumably causes a loss of the secretory DMP1 but does not affect the nuclear translocation of DMP1, shows a typical rachitic tooth defect. Furthermore, we generated transgenic mice expressing (NLS)DMP1, in which the endoplasmic reticulum (ER) entry signal sequence of DMP1 was replaced by a nuclear localization signal (NLS) sequence, under the control of a 3.6 kb rat type I collagen promoter plus a 1.6 kb intron 1. We then crossbred the (NLS)DMP1 transgenic mice with Dmp1 null mice to express the (NLS)DMP1 in Dmp1-deficient genetic background. Although immunohistochemistry demonstrated that (NLS)DMP1 was localized in the nuclei of the preodontoblasts and odontoblasts, the histological, morphological and biochemical analyses showed that it failed to rescue the dental and periodontal defects as well as the delayed tooth eruption in Dmp1 null mice. These data suggest that the full-length DMP1 plays no apparent role in the nucleus during odontogenesis.
Animals ; Cell Nucleus ; genetics ; Codon, Initiator ; genetics ; Collagen Type I ; genetics ; Endoplasmic Reticulum ; genetics ; Extracellular Matrix Proteins ; genetics ; Familial Hypophosphatemic Rickets ; genetics ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Humans ; Introns ; genetics ; Methionine ; genetics ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutation ; genetics ; Odontoblasts ; cytology ; Odontogenesis ; genetics ; Periodontal Diseases ; genetics ; Periodontal Ligament ; pathology ; Phosphoproteins ; genetics ; Promoter Regions, Genetic ; genetics ; Tooth Abnormalities ; genetics ; Tooth Eruption ; genetics ; Transcription Factors ; genetics ; Transgenes ; genetics ; Valine ; genetics ; Young Adult

OBJECTIVEIn the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo.

METHODSHuman cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time.

RESULTSWe observed that treatment with pristimerin, at as low as 1 μmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg·d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes.

CONCLUSIONThese studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

Animals ; Cell Line ; Dependovirus ; genetics ; physiology ; Gene Expression ; drug effects ; Genetic Therapy ; Genetic Vectors ; genetics ; physiology ; Hepatocytes ; metabolism ; virology ; Humans ; Liver ; cytology ; metabolism ; virology ; Mice ; Transgenes ; drug effects ; Triterpenes ; pharmacology

How to reduce immune response is an unprecedented challenge for rAAV gene medicine. Recent studies suggested that lowering dosage of the vector used could reduce immune response caused by rAAV gene medicine. Nevertheless, it would also decrease the transgene expression, leading to failure of gene treatment. It is therefore important to take appropriate steps to maintain high gene expression level and pharmacodynamic, while the dosage of rAAV used is reduced. Here, steps to enhancing gene therapy, such as optimization of the administration, reconstruction of the viral vector and selection of the promoter, are discussed in order to achieve maximum outcome.
Animals ; Dependovirus ; genetics ; immunology ; Dose-Response Relationship, Drug ; Gene Dosage ; Gene Transfer Techniques ; Genetic Therapy ; Genetic Vectors ; administration & dosage ; genetics ; immunology ; Humans ; Recombination, Genetic ; Transgenes ; genetics