Direct Gene Transfer Research Articles

Adeno-associated virus (AAV) 6 provides efficient gene transfer upon intramyocardial injection in vivo

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Innovation Council Eurostars Background Cardiac gene transfer utilizing adeno-associated virus (AAV) is a promising approach for the treatment of various cardiovascular diseases. Intramyocardial injection is an efficient delivery route, and is particularly relevant for ischemic disease or pacemaker and conduction system abnormalities, where local treatment is favoured. However, which AAV variants provide efficient cardiac gene transfer after intramyocardial injection remains to be tested. Aim This study aims to compare several cardiotropic AAV variants in order to identify their relative potency in the setting of direct intramyocardial injection. Methods A green fluorescent protein (GFP)-coding sequence was packaged in the two benchmark AAV capsids, AAV6 and AAV9, and a selection of recently reported AAV9 capsid variants which are named MyoAAVs. These AAV vectors were added to neonatal rat ventricular cardiomyocytes (NRVM) and their transduction efficiency was evaluated by flow cytometry and quantitative PCR. AAV6, AAV9 and MyoAAV4A vectors were then delivered into the left ventricle of FVB mice via intramyocardial injection. AAV6 and AAV9 carrying GFP in a different expression cassette were also intramyocardially injected in FVB and Bl6 mice. Direct fluorescence, transgene mRNA, and viral genomes were quantified to determine the transduction efficiency 4 weeks post injection. Picrosirius red staining was used to assess whether AAV-mediated GFP delivery causes cardiac fibrosis. We further studied GFP encoding AAV6 and AAV9 vectors upon intramyocardial injection into the left ventricle of pigs and determined the transduction efficiency by quantitative PCR. Results NRVM transduction experiment revealed that among the tested capsid variants, AAV6 is the most efficient in transducing cardiomyocytes, followed by MyoAAV4A. In mice, AAV6 is most effective in transducing myocardium upon intramyocardial injection, resulting in approximately 10-fold higher transgene expression than that obtained with AAV9 vector and 2-fold higher than that with MyoAAV4A vector. Histology revealed no vector-related cardiac fibrosis. The use of different expression cassettes and animal strains consistently showed that AAV6 outperforms AAV9 in cardiac transduction efficiency. Transgene expression in liver was only observed in Bl6 mice injected with AAV9. In porcine myocardium, AAV6 led to robust transgene expression after intramyocardial injection and this expression level is higher than that of AAV9. Conclusion Among the AAV variants tested, AAV6 appears to be the most efficient for cardiac transduction by intramyocardial injection. These results suggest that AAV6 represents an attractive vector for direct cardiac gene transfer and for future capsid engineering in the context of local transgene delivery.

Unrevealing the potential of aquatic macrophytes for phytoremediation in heavy metal-polluted wastewater

Aquatic macrophytes including Eichhornia crassipes, Pistia stratiotes, and Lemna minor have shown promise in phytoremediation for wastewater treatment and heavy metal removal. This approach may reduce pollution. However, plant metabolic rate and ability to break down organic compounds or tolerate heavy metals are limited. Biotechnology can significantly improve these issues. Scientists can alter plants to improve phytoremediation using genetic engineering. Direct gene transfer allows certain genes to be introduced into plants, giving them advantages. Introducing genes involved in metal absorption, transport, and detoxification into plants may increase their ability to store and survive heavy metals. Introducing genes that encode enzymes that break down organic pollutants can also improve decomposition. Transgenic plants with improved metabolic pathways, biomass yield, and stress tolerance have been created using biotechnology. These advances benefit phytoremediation. Selecting acceptable plant species for genetic modification is crucial. Aquatic macrophytes can produce a lot of biomasses, grow quickly, and absorb nutrients, making them excellent for genetic engineering in phytoremediation. In general, biotechnology can improve phytoremediation by overcoming plant capacity restrictions. The ecological and regulatory effects of introducing genetically modified organisms into the environment must be considered. The safe and sustainable use of genetically modified plants for phytoremediation requires collaboration between biotechnologists, environmental scientists, and legislators.

Pre-clinical delivery of gene therapy products to the cerebrospinal fluid: challenges and considerations for clinical translation.

While the majority of gene therapy studies in neurological indications have focused on direct gene transfer to the central nervous system (CNS), there is growing interest in the delivery of therapeutics using the cerebrospinal fluid (CSF) as a conduit. Historically, direct CNS routes-of-administration (RoAs) have relied on tissue dynamics, displacement of interstitial fluid, and regional specificity to achieve focal delivery into regions of interest, such as the brain. While intraparenchymal delivery minimizes peripheral organ exposure, one perceived drawback is the relative invasiveness of this approach to drug delivery. In this mini review, we examine the CSF as an alternative RoA to target CNS tissue and discuss considerations associated with the safety of performing such procedures, biodistribution of therapeutics following single administration, and translation of findings given differences between small and large animals. These factors will help delineate key considerations for translating data obtained from animal studies into clinical settings that may be useful in the treatment of neurological conditions.

A Review on Understanding the Effects and Mechanisms of Salinity Tolerance in Rice (Oryza sativa L.)

Salinity, along with drought, is one of the key abiotic stressors that has posed a danger to the advancement and evolution of cereal crops like rice and wheat. Water shortage and a lack of irrigation water availability are the main causes of salty soil formation. Rice is salt sensitive and glycophyte, wheat is moderately salt tolerant. Wild tolerant cultivars like Oryza coarctata and Oryza alta are more tolerant than traditional cultivars such as Pokkali and Nona Bokra in rice. Salt stress affects crop plants’ processes like ionic imbalance, osmotic and oxidative stress. Na+ should be low in the shoots of the plant which is restricted by various transporters in the cell membrane of the roots in soil. High K+ & Na+/K+ homeostasis should be maintained. Many RILs and NILs have been developed which acts as a donor for salinity tolerant genes. FL478 is a recombinant inbred line in which candidate genes are situated in the Saltol region of chromosome 1 region which is obtained by a cross between Pokkali x IR29. Increase in world’s population, rice output must be increased by at least 25% by 2030 and 50% by 2050.Salinity stress is a polygenic character which involves several genes works in harmony. For evolution of salinity tolerant cultivars, we need to access the physiological, biochemical genetic responses of the crop plant which helps in transfer of candidate genes from donor parents to elite high yielding salt sensitive cultivars. Especially in rice salt tolerant mechanisms like, Ion equilibrium regulation, Adjustment of osmotic potential, Reduction of ROS, Nutrient disequilibrium, and Regulation of PGRs. Conventional, MABC, MAS and direct gene transfer by transgenic methods. This review paper's main objective is to understand the mechanisms of the crop plants to salinity effects and development of salt tolerant cultivars by modern approaches which fulfill the food scarcity of staple food crops with increasing population.

Past, present and future prospect on microinjection gene transfer in aquaculture

Microinjection is arguably the most direct and reliable gene transfer method to date. Since the development of the first transgenic fish in 1984, microinjection continues to be the most commonly used gene transfer method in aquaculture. Microinjection is used to inject cells, nucleic acid, gene constructs, recombinant DNA, and morpholino oligonucleotides into fish cells, embryos, and larvae. Despite the development of various gene transfer methods, microinjection is preferable. Continuous improvement in micromanipulation tools and micro capillary further develop microinjection method. This review describes the past and present use of microinjection gene transfer methods for genetic manipulation and genetic improvement in fisheries and aquaculture, especially in fish transgenesis. Furthermore, we discuss the future prospects of microinjection method in tandem with recombinant DNA technology and CRISPR-Cas9 and its application in the aquaculture industry.

Intra-articular injection of rAAV-hFGF-2 ameliorates monosodium iodoacetate-induced osteoarthritis in rats via inhibiting TLR-4 signaling and activating TIMP-1

Osteoarthritis (OA) is a chronic debilitating degenerative disorder leading to structural, and functional anomaly of the joint. The present study tests the hypothesis that overexpression of the basic fibroblast growth factor (FGF-2) via direct rAAV-mediated gene transfer suppresses monosodium iodoacetate (MIA)-induced knee OA in rats relative to control (reporter rAAV-lacZ vector) gene transfer by intra-articular injection. Rats were treated with 20 μl rAAV-hFGF-2 on weekly basis; on days 7, 14, and 21 after single intra-articular injection of MIA (3 mg/50 μl saline). FGF-2 reduced knee joint swelling and improved motor performance and muscle coordination as evidenced by increased distance travelled, mean speed, rearing frequency in open field test (OFT) as well as fall-off latency in rotarod test together with reduced immobility time in OFT. Moreover, FGF-2 attenuated MIA-related radiological and histological alterations. Indeed, FGF-2 decreased knee joint inflammatory biomarker as demonstrated by reduced mRNA expression of toll like receptor (TLR)-4 and its downstream mediators such as tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β) and high motility group box (HMGB) 1. In parallel, FGF-2 attenuated knee joint degradation biomarkers as reflected by the downregulation of ADAMTS-5 mRNA expression and matrix metalloproteinase 13 (MMP-13) content together with the up-regulation of tissue inhibitor of metalloproteinase (TIMP)-1 mRNA expression. These findings suggest a potential therapeutic role for FGF-2 against MIA-induced knee OA in rats via inhibition of TLR4 signaling and activating TIMP-1, resulting in down-regulation of ADAMTS-5 and MMP-13.

Neurologic Recovery in MPS I and MPS II Mice by AAV9-Mediated Gene Transfer to the CNS After the Development of Cognitive Dysfunction.

The mucopolysaccharidoses (MPS) are a group of recessively inherited conditions caused by deficiency of lysosomal enzymes essential to the catabolism of glycosaminoglycans (GAG). MPS I is caused by deficiency of the lysosomal enzyme alpha-L-iduronidase (IDUA), while MPS II is caused by a lack of iduronate-2-sulfatase (IDS). Lack of these enzymes leads to early mortality and morbidity, often including neurological deficits. Enzyme replacement therapy has markedly improved the quality of life for MPS I and MPS II affected individuals but is not effective in addressing neurologic manifestations. For MPS I, hematopoietic stem cell transplant has shown effectiveness in mitigating the progression of neurologic disease when carried out in early in life, but neurologic function is not restored in patients transplanted later in life. For both MPS I and II, gene therapy has been shown to prevent neurologic deficits in affected mice when administered early, but the effectiveness of treatment after the onset of neurologic disease manifestations has not been characterized. To test if neurocognitive function can be recovered in older animals, human IDUA or IDS-encoding AAV9 vector was administered by intracerebroventricular injection into MPS I and MPS II mice, respectively, after the development of neurologic deficit. Vector sequences were distributed throughout the brains of treated animals, associated with high levels of enzyme activity and normalized GAG storage. Two months after vector infusion, treated mice exhibited spatial navigation and learning skills that were normalized, that is, indistinguishable from those of normal unaffected mice, and significantly improved compared to untreated, affected animals. We conclude that cognitive function was restored by AAV9-mediated, central nervous system (CNS)-directed gene transfer in the murine models of MPS I and MPS II, suggesting that gene transfer may result in neurodevelopment improvements in severe MPS I and MPS II when carried out after the onset of cognitive decline.

Notch Pathway Driven Delayed Dedifferentiation of Liver Sinusoidal Endothelium Impacts Liver-Directed Gene Transfer in Hemophilia-a Mice

Background: Hemophilia A is an X-linked recessive bleeding disorder that affects 1 in 5000 males and is caused by procoagulant factor VIII deficiency. Affected people are at danger of spontaneous bleeding into organs, which can be fatal and lead to persistent damage. Current therapy includes intravenous infusion of FVIII protein concentrate which is costly, inconvenient, and does not fully prevent bleeding. As a result of recent advancements in liver-directed gene transfer, gene therapy based innovative strategies for treating hemophilia have emerged. The Factor VIII null mouse (F8TKO mice) has been effective in understanding the disease pathogenesis as well as the development of liver directed novel gene therapy techniques to treat hemophilia. FVIII is predominantly produced in the liver sinusoidal endothelial cells (LSECs). Thus, liver directed adenoviral and retroviral vectors have been studied by several groups to understand the gene delivery method in hemophilia. Methods: In this study we evaluated the stability and efficacy of liver driven gene transfer mechanism in F8TKO mouse using recombinant adeno associated virus (AAV)8 vector. AAV8-TBG-GFP was administered through IP and IV, and GFP expression was analyzed in the liver 15- and 30-days post injection. Results: Immunofluorescence of GFP at day 15 and day 30 post injection revealed significantly fewer GFP+ cells (p=0.04) in the liver of F8TKO compared with control mice post AAV8-GFP injection. This was further validated by ELISA assays and western blot for GFP. Moreover, Caspase-3 staining revealed significant enrichment (p=0.05) in the liver of F8TKO mice compared with control mice after AAV8-GFP administration at both the 15- and 30-day time points suggestive of increased LSEC apoptosis post AAV8-GFP injection. To further confirm apoptosis of LSECs, we next used liver intravital imaging to visualize LSECs in F8TKO mice at baseline and upon AAV8-GFP administration. TXR-dextran and AF488-anti-CD31 antibody were IV administered to visualize hepatic blood flow and LSECs, respectively. At baseline, both control and F8TKO mice showed normal blood flow and enriched expression of AF488-anti-CD31. However, there were fewer AF488-anti-CD31+endothelial cells in F8TKO than control mice at day 15 post-AAV8-GFP treatment (p=0.01), suggestive of apoptosis of LSECs in F8TKO mice post-AAV8-GFP administration. Scanning electron microscopy revealed dramatic reduction (p=0.01) in endothelial fenestration in the liver of F8TKO as compared with control mouse liver. The absence of fenestrae has been associated with LSEC capillarization, which promotes basement membrane formation while decreasing permeability. Remarkably, we found significant upregulation of LSEC markers associated with capillarization, including CD31, vascular endothelial growth factor (VEGF), ICAM1 (Intercellular Adhesion Molecule-1), stabilin2, ID1 (Inhibitor of DNA Binding-1), Gata4, and Ehd3 (EH Domain Containing-3) in F8TKO mice by qRT-PCR) and western blot. Mechanistically we show significant upregulation of Notch pathway components (Notch 1, 3, and Jagged 2) by qRT-PCR and western in the liver of F8TKO mice. IHC analysis showed significant upregulation of notch 3 early in development in F8TKO mice leading to pseudocapillarization of LSECs. Moreover, we also show that notch activation regulates nitric oxide/soluble guanylyl cyclase signaling by upregulating downstream targets (NOS-1, 2, 3, PDE1a, b, c and 10B) in LSECs which directly controls the development of LSEC fenestration. Conclusion: In conclusion, we demonstrate molecular characterization of LSEC-mediated liver-directed gene transfer in a null hemophilia A mouse model, which is critical for understanding the efficacy and stability of AAV-FVIII gene-based hemophilia treatment. Work is currently underway to understand how absence of FVIII can affect the LSECs mediated hepatic gene transfer in Hemophilia-A mice and how modulation of Notch and nitric oxide/sGC signaling can manipulate LSEC fenestration and improve the efficiency of liver directed gene transfer.

Reshaping the Tree of Life: ecological implications of evolution in the Anthropocene

Novel in their scope and intensity, human‐mediated changes in genetic diversity through directed gene transfer technologies and longer standing human‐driven selective pressures, such as land‐use change, species introductions, mass extinctions, and broad application of antibiotics, are combining to reorganize mechanisms of evolution. The evolutionary consequences of anthropogenic change can be observed across levels of biological organization and are influencing the rate of micro‐ and macroevolutionary changes, as well as feedback among them. This may have large‐scale effects on the provisioning of ecosystem services, food security, and human health. Here, we summarize several of the ecological implications of human modification of evolutionary dynamics, focusing specifically on emerging molecular technologies, to highlight some of the challenges in predicting subsequent changes in the world’s ecosystems.

A Dedifferentiated Sinusoidal Endothelium Impacts Liver-Directed Gene Transfer in Hemophilia-a Mice

Hemophilia A is an X-linked recessive bleeding disorder that affects 1 in 5000 males and is caused by procoagulant factor VIII deficiency. Affected people are at danger of spontaneous bleeding into organs, which can be fatal and lead to persistent damage. Current therapy includes intravenous infusion of FVIII protein concentrate which carries the danger of transmitting blood-borne diseases. As a result of recent advancements in liver-directed gene transfer, gene therapy based innovative strategy for treating hemophilia has emerged. In patients with severe hemophilia B, intravenous infusion of an adeno-associated viral (AAV) vector encoding factor IX (FIX) under the control of a liver-directed promoter resulted in expression of FIX for a considerable period of time. In hemophilia-A patients, gene treatment utilizing AAV vectors has demonstrated to be less effective than Hemophilia B due to the size of the F8 coding sequence and the decreased release of FVIII protein. Among other concerns high immunogenicity of FVIII with 25-30% of hemophilia A patients forming inhibitors and overexpression of FVIII in hepatocytes triggering a cellular stress response are significantly challenging. A phase 1 clinical trial is now being conducted to examine the AAV8 induced liver directed gene expression strategy to circumvent these challenges.The Factor VIII null mouse has been effective in understanding the disease pathogenesis as well as the development of liver directed novel gene therapy techniques to treat hemophilia. FVIII is predominantly produced in the liver. Thus, liver directed adenoviral and retroviral vectors have been studied by several groups to understand the gene delivery method in hemophilia. A few of these studies have shown limited effectiveness in hemophilia animal models. Although the coagulation anomaly seen in hemophilia murine model was completely repaired immediately after liver directed adenovirus-mediated treatment, the effect was transient. Additionally, adeno associated virus (AAV8)-FVIII overexpression has been associated with increased cellular stress. In this study we evaluated the stability and efficacy of liver driven gene transfer mechanism in FVIII null mouse using recombinant AAV8 vector. Recombinant AAV8 vector delivered through the systemic circulation successfully transduces to target tissues via passing through the permeable barrier of sinusoidal endothelial cell. The bidirectional passage through sinusoidal endothelial cell is mainly supported by the presence of discontinuous fenestrated endothelium. Remarkably, we found that liver directed gene transfer was significantly delayed in FVIII null mice. Using quantitative liver intravital imaging we found that upon AAV8-GFP administration liver sinusoidal endothelial cells shows increased apoptosis. Moreover, structural analysis of the liver sinusoidal endothelial cells using intravital and electron micrograph imaging showed significant structural functional difference in liver sinusoidal endothelial cells of FVIII KO mouse. Work is currently underway to understand how absence of FVIII can affect the LSECs. In conclusion, detailed molecular characterization of LSEC-mediated liver directed gene transfer in a hemophilia mouse model is critical for understanding the efficacy and stability of gene-based hemophilia treatment. DisclosuresSundd: Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring Inc: Research Funding.

A Comprehensive Review of Vaccines against Covid-19

A Comprehensive Review of Vaccines against Covid-19

A positive experience in applying the biolistic approach to potato varieties Aksor and Nevskiy.

The method of biological ballistics (biolistic transformation, genetic bombardment) of plants is one of the most modern methods used for direct gene transfer into plant cells. The main advantages of this method include the ability to simultaneously incorporate several target genes into the plant genome, carry out transfer without unnecessary agrobacterial parts and plasmid DNA sequences, and the short time needed to produce transgenic cells. For different plant objects, the efficiency of obtaining transgenic plants by the ballistic method varies from 1 to 3 %. For potato plants, the transformation efficiency is quite low at the moment and the selection of optimal conditions for biolistics is one of the pressing issues of practical biotechnology. This article presents a successful experience of introducing two genes of interest into two potato varieties using the biolistic approach. The results of biolistic transformation experiments are presented for two types of explants: potato internodes and calli of the varieties Aksor and Nevskiy. Of the 862 explants used for transformation, 56 regenerated plants were obtained. PCR screening of transformants revealed one plant with the insertion of the chitinase gene, one with the insertion of the endo-β-1,3-glucanase gene, and co-transformation by both genes was confirmed in four regenerants. The average transformation efficiency for potato explants was 0.7 %. A high number of regenerants (56) as opposed to a low number of transformants (6) reflects an attempt to increase the number of regenerants by using a lower concentration of the selective agent (kanamycin). Although this approach requires more effort, it can be used to produce potato lines with integrated genes of interest for further use in crop breeding. The lines of potato obtained in the current study by introducing two genes associated with the plant response to fungal pathogens will be further assessed for their resistance to fungal diseases and, if successful, will be used in potato crop breeding.

RAAV-Mediated Human FGF-2 Gene Therapy Enhances Osteochondral Repair in a Clinically Relevant Large Animal Model Over Time In Vivo

Background: Osteochondral defects, if left untreated, do not heal and can potentially progress toward osteoarthritis. Direct gene transfer of basic fibroblast growth factor 2 (FGF-2) with the clinically adapted recombinant adeno-associated viral (rAAV) vectors is a powerful tool to durably activate osteochondral repair processes. Purpose: To examine the ability of an rAAV-FGF-2 construct to target the healing processes of focal osteochondral injury over time in a large translational model in vivo versus a control gene transfer condition. Study Design: Controlled laboratory study. Methods: Standardized osteochondral defects created in the knee joints of adult sheep were treated with an rAAV human FGF-2 (hFGF-2) vector by direct administration into the defect relative to control (reporter) rAAV-lacZ gene transfer. Osteochondral repair was monitored using macroscopic, histological, immunohistological, and biochemical methods and by micro–computed tomography after 6 months. Results: Effective, localized prolonged FGF-2 overexpression was achieved for 6 months in vivo relative to the control condition without undesirable leakage of the vectors outside the defects. Such rAAV-mediated hFGF-2 overexpression significantly increased the individual histological parameter “percentage of new subchondral bone” versus lacZ treatment, reflected in a volume of mineralized bone per unit volume of the subchondral bone plate that was equal to a normal osteochondral unit. Also, rAAV-FGF-2 significantly improved the individual histological parameters “defect filling,”“matrix staining,” and “cellular morphology” and the overall cartilage repair score versus the lacZ treatment and led to significantly higher cell densities and significantly higher type II collagen deposition versus lacZ treatment. Likewise, rAAV-FGF-2 significantly decreased type I collagen expression within the cartilaginous repair tissue. Conclusion: The current work shows the potential of direct rAAV-mediated FGF-2 gene therapy to enhance osteochondral repair in a large, clinically relevant animal model over time in vivo. Clinical Relevance: Delivery of therapeutic (hFGF-2) rAAV vectors in sites of focal injury may offer novel, convenient tools to enhance osteochondral repair in the near future.

305 In vivo correction of recessive dystrophic epidermolysis bullosa (RDEB) by direct cutaneous COL7A1 gene replacement: Results of a phase 1-2 trial

The use of direct gene transfer to correct genetic skin disease has been a longstanding goal in the gene therapy field. Here we demonstrate correction of the genetic disorder RDEB by the application of a replication defective herpes simplex virus-1 vector encoding the type VII collagen (C7) gene COL7A1, bercolagene telserpavec (BVEC), directly to skin via a phase 1-2 clinical trial. This trial was an intra-patient comparison of recurrent and chronic wounds administered with either topical BVEC or topical placebo applied every other day until healing.

Review: Some Molecular Breeding Issues at Genome Level in Fruit Species

The traditional plant breeding studies preferred for centuries are still widely used. Human beings started their first selection studies within the scope of plant breeding by making use of wild plant products in order to meet their nutritional needs. Later, the first growers have discovered foreign pollination to increase the yield of some agricultural crops. Thus, they could make improvements in the vegetative characteristics of new offspring individuals by hybridization by artificial pollination in different parent plants. With a better understanding of the science of genetics, crossbreeding studies were also important in order to obtain new individuals with desired features. Plant breeding studies haave improved further in the 20th century, and the mechanisms for how to obtain new agricultural varieties with superior qualifications that will keep the desired characteristics are better understood. The rapid developments in the fields of quantitative genetics, mutation breeding, polyploidy breeding and plant tissue culture studies have formed the foundations of today's plant breeding issues. In parallel with the advances in gene cloning, direct gene transfer studies to plant tissues have also been successful. Gel, hybridization and expression systems; cell imaging by light and electron microscopy; With advances in molecular techniques such as high-density microarrays and sequencing, various areas of 'omics' such as genomics, transcriptomics, proteomics, metabalomics and phenomics have been contributed. In the 2000s, plant breeders focused more on genomic studies. Plant breeding studies are now planned with intense interest on genome-wide association studies and genome editing (genome design) studies. The aim of this study is to give brief information about some molecular breeding issues that are being carried out at the genome level in the breeding of perennial fruit species. Molecular maps in fruit breeding, selection approaches at the molecular level and the purpose of creating genetic maps based on marker-character relationship are briefly explained by presenting examples. Although there are many documents about fruit breeding issues in different titles and in very detailed contents around the world, it is important to present a summary of these issues with Turkish narration for those interested in fruit breeding in our country and to share which fruit breeding subjects are studied. Keywords: Fruit breeding, Characterization, Molecular maps, Selection, Association maps DOI: 10.7176/JSTR/6-08-08

Gene expression variability between randomly and targeted transgene integration events in tobacco suspension cell lines

Genome editing tools such as zinc-finger nucleases provide novel strategies for genetic manipulation in plants. Unlike agrobacterium-mediated or direct gene transfer, which introduce genes randomly into the genome and thereby potentially resulting in high variation of gene expression, the targeted gene addition provides predictable integration of DNA sequences into a specified location of the plant genome. We investigated whether various independent cell lines that all contain a transgene placed in the same genomic locus by zinc-finger nuclease-mediated homologous recombination (HR) would yield a more reproducible and homogeneous level of expression compared to integration events generated via agrobacterium-mediated transformation at random sites. The variance of gene expression of targeted HR events and random integration events was analyzed in Nicotiana tabacum L cv. Bright Yellow 2 (BY-2) suspension cells by measuring protein amount produced from the transgene by flow cytometry, thus providing the first report on positional effects of marker gene expression in a quickly proliferating plant suspension cell line. Marker protein levels of targeted HR and single-copy random events covered a similar range; however, the uniformity of protein expression in a given cell line was significantly higher in targeted events than in lines with randomly inserted transgene; the same is true for the overall viability of protoplasts from HR lines. In conclusion, using targeted insertion into a qualified locus of a well-characterized line leads to more reliable results than random insertion into the genome.

Target-specific gene delivery in plant systems and their expression: Insights into recent developments

In order to improve crop plants in terms of their yield, drought resistance, pest resistance, nutritional value, etc., modern agriculture has relied upon plant genetic engineering. Since the advent of recombinant DNA technology, several tools have been used for genetic transformations in plants such as Agrobacterium tumefaciens, virus-mediated gene transfer, direct gene transfer systems such as electroporation, particle gun, microinjection and chemical methods. All these traditional methods lack specificity and the transgenes are integrated at random sites in the plant DNA. Recently novel techniques for gene targeting have evolved such as engineered nucleases such as Zinc Finger Nucleases, Transcription Activator like effector nucleases, Clustered regular interspaced short palindromic repeats. Other advances include improvement in tools for delivery of gene editing components which include carrier proteins, and carbon nanotubes. The present review focuses on the latest techniques for target specific gene delivery in plants, their expression and future directions in plant biotechnology.

Optimizing the particle bombardment conditions in cell suspension cultures of Nicotiana tabacum and expression of the recombinant antihypertensive amarantin

The first successful attempt to produce transgenic tobacco cell suspensions expressing a modified version of amarantin, through direct gene transfer using particle gun is described. The physical parameters for transformation were optimized using the gene encoding β-glucuronidase (gusA) as the reporter gene. Statistical analysis revealed that helium pressure, target distance, and osmotic pretreatment had significant influence on transformation efficiency. Of the different variables evaluated, a combination of sorbitol 0.5 M pretreatment, shooting distance of 7 cm and firing pressure of 900 psi were found ideal. Selection of putative transgenic calli was done on Hygromicine-selection medium. Presence of the transgene was confirmed using histological glucuronidase assay and PCR analysis. Accumulation and stable expression of the recombinant amarantin was detected using Western blot analysis. Transformation efficiency in terms of biochemical GUS assay was of 60%. The present results demonstrate the possibility of incorporating and expressing transgene encoding recombinant proteins of interest, in tobacco cell suspension through biolistic delivery system.

MRNA: A Novel Avenue to Antibody Therapy?

First attempts to use exogenous mRNA for protein expression invivo were made more than 25 years ago. However, widespread appreciation of invitro transcribed mRNA as a powerful technology for supplying therapeutic proteins to the body has evolved only during the past few years. Various approaches to turning mRNA into a potent therapeutic have been developed. All of them share utilization of specifically designed, rather than endogenous, sequences and thorough purification protocols. Apart from this, there are two fundamental philosophies, one promoting the use of chemically modified nucleotides, the other advocating restriction to unmodified building blocks. Meanwhile, both strategies have received broad support by successful mRNA-based protein treatments in animal models. For such invivo use, specifically optimized mRNA had to be combined with potent formulations to enable efficient invivo delivery. The present review analyzes the applicability of mRNA technology to antibody therapy in all main fields: antitoxins, infectious diseases, and oncology.

Adenoviral delivery of adiponectin ameliorates osteogenesis around implants in ovariectomized rats.

Adiponectin (APN) has been reported to promote bone formation. However, it is difficult to utilize a conventional method that administers sufficient APN to the implant site. The present study investigated the efficacy of an APN transgene to accelerate the implant osseointegration in ovariectomized (OVX) rats. In vitro, bone marrow stromal cells were transduced with reconstructed adenovirus (Ad-APN-EGFP) and osteoclast precursor RAW264.7 cells were co-cultured with the conditioned medium secreted by transduced bone marrow stromal cells. Tartrate-resistant acid phosphatase staining and bone slice resorption assay were performed to evaluate the activity of osteoclastogenesis. In vivo, Ad-APN-EGFP was administered into the bone defect prior to implant placement in OVX rats. At 7 and 28days post implantation, the femurs were harvested and prepared for a real-time reverse transcriptase-polymerase chain reaction, hemotoxylin and eosin staining, immunohistochemical staining, micro-computed tomography analysis and biomechanical testing. The results showed the formation and function of osteoclasts were significantly suppressed in vitro. Successful transgene expression was confirmed, and a significant increase of OCN, Runx2 and ALP expression was detected in the Ad-APN-EGFP group in vivo. Interestingly, we also found that the overexpression of APN decreased the expression level of potent adipogenic transcription factors such as PPARγ2 and C/EBP-α. At 28days after implantation, the Ad-APN-EGFP group revealed a significantly increased osseointegration and implant stability in OVX rats compared to the control groups (Ad-EGFP and PBS groups). APN via direct adenovirus-mediated gene transfer could ameliorate osseointegration surrounding titanium implants in OVX-related osteoporosis rats. Furthermore, it may be an effective strategy for promoting bone regeneration under osteoporotic conditions.