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Lentiviruses are considered one of the most effective recombinant viruses for gene transfer to mammalian cells and tissues. In this study, the potential of HIV-1-based lentiviral vector to deliver transgenes into avian cells was examined. We co-transfected human embryonic kidney cell line HEK-293T with three lentivirus vectors called transfer, packaging and envelope vectors. We collected the supernatant from transfected cells 24 and 48 hours post-transfection and filtered them immediately. Then we subjected the filtered supernatant to Amicon protein columns for concentration purposes. Centrifugation removed a larger part of the supernatant presumably free of viruses and left behind a small volume of darken solution full of virions. We thereby produced a 500-µl-volume of virus stock. Various dilutions of this stock were added to chicken liver cell line LMH. The initial sign of infection appeared within 48 hours and by 96 hours post-infection 100% the LMH cells positively expressed transgenes. Our results indicated that the human HIV-1-based lentivirus vectors are capable of transducing and transferring foreign genes into chicken cells. Given the need for a high-titer virus stock for successful target cell transduction, our results indicate that the filtration method of virus concentration is able to produce high virus titer and is cost-effective and less time consuming than ultracentrifugation or other traditional methods.

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Lentiviruses are considered one of the most effective recombinant viruses for gene transfer to mammalian cells and tissues. This study comprises of two essential parts: (1) evaluation of efficiency of protein purification columns in concentration of recombinant lentiviruses, and (2) production of recombinant lentiviruses carrying GDNF coding sequences. In part (1) we co-transfected human embryonic kidney cell line HEK-293T with three lentivirus vectors called transfer (carrying either GFP or Jred), packaging and envelope vectors. After a filtration step, we applied the supernatant from transfected cells to Amicon protein columns for concentration purposes. Centrifugation removed 99% of the supernatant and left behind 500-µl-volume of solution full of virions. We thereby produced a of virus stock. Various dilutions of this stock were added to HEK-293T cells that produced up to 100% infected cells positively expressing transgenes. To examine whether the removed supernatant (overflow) has any trace of infective virus by chance, we also used dilutions of the overflow for infection and observed no sign of eGFP or Jred expression. Given the need for a high-titer virus stock for successful target cell transduction, our results indicate that our filtration method of virus concentration is able to produce high virus titer and is cost-effective and less time consuming than previous methods. In part (2), due to the importance of neurotrophic factor GDNF in differentiation and neuroprotection as well as in therapy of neurodegenerative disorders, we ligated GDNF coding sequence into the lentivirus backbone in the second phase of our study. We applied the same method outlined above to produce high-titer recombinant viruses. Following infection of human astrocytoma cells with this virus stock, we detected 3-fold increase in GDNF mRNA expression using RT-PCR. Lentiviruses carrying GDNF can therefore be generated at high titer using the column method and applied for differentiation and neuroprotection studies.

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Stem cell therapy has been introduced as an innovative and promising treatment in Ischemic diseases. Mesenchymal stem cells are considered for cell therapy to some extent due to their immunemodulatory, differentiation potential, feasibility of isolation and proliferation properties. Stem cells, after transplantation, often encounter harsh and hypoxic environment in ischemic tissues, which leads to cell death and decreased therapeutic efficiency. On the other hand, the fate of stem cell viability and differentiation  is still an ambiguous issue in cell therapy regenerative medicine. To overcome this problem, Hypoxic/Ischemic preconditioning has been reported as a powerful tool with beneficial effects on cell survival. The reported master regulator in this process is a transcription factor known as HIF-1α. This study aimed to over-express HIF-1α in mesenchymal stem cells along with eGFP by using lenti viral vectors. Bisistronic expression of eGFP and HIF-1α provides the possibilities of tracking the transplanted cells and mimicking the hypoxic conditions for genetically modified stem cells for future animal model studies.  

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Escherichia coli is a Gram-negative bacterium, the second most common bacteria in the intestine and the main indicator of urban water pollution, is the most common cause of urinary tract infection and also is one of the main factors in food poisoning and diarrhea. Drug resistance of this bacterium to antibiotics is a global challenge. Horizontal gene transfer (HGT) is the movement of genetic material between unicellular and other gene transfer pathways which is an important factor in the evolution of many organisms, antibiotic resistance in bacteria, gene function. Antibiotic resistance in E. coli can be transferred to another species of bacteria through HGT mechanisms. Today, Bioinformatics methods have been used to understand of gene transfer from HGT mechanism. In this study, we used bioinformatics tools such as PredictBias, ACLAME, Mobil Genetic Elements (MGEs) PAI-ID, and Alien_Hunter in order to genes analysis that related from antibiotic resistance in E. coli. Bioinformatics and MIC assays result show that from 26 to 30 genes have been identified in all safthwers. Most of genes that identified show over 50 percent of GC content. put P gene with 178, blaCMY with 62, BlaTEM with 43, and aac-6 with 66 homology in the PredictBias website identified. Also in the ACLAME website, mob (A-C) and rep (A-C) gene family are highest number of horizontal gene transfer from infection bacteria strain. Those cluster genes are the highest resistant of laboratory tests which carries resistant genes such as  blaSHV and blaCHV on the blaCMY plasmid.       

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Wild pepper (Capsicum frutescens L.) could be a source of variation to improve cultivated pepper due to its unique traits with adapting challenges caused by adversity. Interspecific hybridization has been used as an effective way of pepper introgression breeding, which transfers genes of interest from wild relatives to cultivated crops. Here, eight fertile hybrids F₁ were produced from pepper (Capsicum annuum L.) and the wild relative (C. frutescens), as female and male, respectively, by interspecific hybridization. Interspecific hybrids were identified using conventional morphological descriptors and SSR molecular markers. The results showed that significant differences in agronomic traits existed among cultivated pepper, wild relatives, and interspecific hybrid F₁. Interspecific hybrid F₁ presented intermediate values, although they were closer to the wild species in most of the agronomic traits. Analysis of SSR markers clearly showed that interspecific hybrid F₁ had bands from the paternal and maternal accessions, which indicated that F₁ hybrid was heterozygous. Our results provide hybrid for breeder to transfer genes of interest from wild relative, C. frutescens, to cultivated pepper, which is an important step for introgression breeding.