PSX-B-16 Recovery of live cells from refrigerated sheep skin after different days of cold storage

Cryopreservation of tissues from domesticated and wild relatives has been suggested to conserve genetic diversity. Ensuring that the tissues have live cells prior to preservation, especially in postmortem tissues, is an essential stem for success. How long cells live after clinical death is not precisely known in animals. The objective of this study was to evaluate the limits of cell survival in sheep skin stored at 4°C postmortem. Ear skin was procured from six random but healthy slaughtered animals and stored at 4°C in the lab. Ten explants (2–3 mm2) were cultured from each animal in DMEM media with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 2.5 µg/mL of fungizone on two 60 mm dishes after 0, 10, 20, 27, 30, 35, 38, 41, 45, 50, 55, 60, 65 and 70 days of storage. Outgrowth of fibroblast-like cells around the explants was scored after 10 days of culture in a CO2 incubator. Results show outgrowth of cells up to 65 days of postmortem storage. Out of 476 explants adhered to dish surface, 374 (78.58%) exhibited outgrowth. The number of outgrowing cells decreased with increasing postmortem storage time interval. To test the differences between cell cultures obtained from postmortem fresh and stored tissues, we established secondary cultures from primary cells of 0-dpm and 65-dpm time points from selected cell lines. Both cultures exhibited similar growth morphology and growth curve, could be cryopreserved with >80% post freezing cell viability, lasted in cultures up to 35 passages, and expressed GFP gene upon transfection with a GFP gene containing plasmid vector. The karyotype analysis of 65-dpm tissue derived cells revealed a normal female karyotype without any genetic aberrations. These results suggest that normal proliferative cells can be recovered from sheep skin up to about 2 months postmortem, if kept refrigerated.

Creation of an inducible vector system based on the rhizobia nodA gene promoter

Background: The possibility of changing the properties of rhizobial bacteria by giving them the ability to regulate the expression of additionally introduced genes into them is an urgent task both for fundamental science and for applied agrobiology, since this will make it possible to obtain microsymbionts with desired properties. An expression construct using the rhizobia regulatory system was created in this work. The rhizobia nodD gene encodes a regulatory protein that, in the presence of plant inducers, flavonoids, activates the transcription of nod-genes involved in the early stages of the formation of legume-rhizobium symbiosis. Materials and methods: A vector construct containing the nodD gene from Rhizobium leguminosarum bv. trifoli under the regulation of its own promoter and the gfp gene under the regulation of the nodA gene promoter from the same rhizobia was obtained. Neorhizobium galegae CIAM 0702 were transformed with the vector construct. Results: It has been shown that in recombinant strains synthetic flavonoids are capable of inducing expression of gfp gene to varying degrees. Conclusion: In the future, the results can be used to obtain rhizosphere microorganisms with a controlled synthesis of growth-stimulating and protective substances.

ANÁLISE DO EFEITO OVICIDA DO dsRNA QUITINA SINTASE NO MOSQUITO Aedes aegypti (DIPTERA: CULICIDAE)

Introdução: O mosquito Aedes aegypti é o principal vetor das arboviroses dengue, chikungunya, Zika e febre amarela. Para que ocorra a produção e a maturação de ovos, as fêmeas do mosquito realizam o repasto sanguíneo, quando ocorre a infecção e transmissão viral. A melhor maneira de evitar a propagação destas doenças é através do controle vetorial. Inseticidas químicos são utilizados no controle, entretanto, ao longo do tempo, os mosquitos adquirem resistência a esses compostos. Neste contexto, o RNA de interferência (RNAi) tem sido uma grande alternativa para o controle. Os genes de quitina sintase (CHSA e CHSB) são considerados excelentes alvos de silenciamento, pois, não são transcritos em vertebrados e plantas. Objetivos: Analisar os efeitos do silenciamento dos genes CHSA e CHSB na oviposição dos mosquitos tratados com dsRNACHS em baixas concentrações. Materiaisemétodos: A produção de dsRNA foi feita in vivo através da transformação de Escherichia coli, cepa HT115 com o plasmídeo L4440, contendo o inserto para a região do sítio catalítico de CHSA e CHSB e GFP (gene não-relacionado para mosquitos), usado como controle. Estas culturas foram lisadas com clorexidina 0,5%. O experimento foi feito em triplicata biológica, utilizando 10 larvas de 4° instar por grupo em 2 mL de água. Foi utilizado, em ambas condições, a concentração 2x10-5 µg/mL de células contendo dsRNACHS ou dsRNAGFP. Os mosquitos adultos sobreviventes foram alimentados com sangue de aves para testar a capacidade de oviposição e a viabilidade dos ovos postos. Resultados: As fêmeas adultas oriundas das larvas tratadas com dsRNACHS tiveram menor postura de ovos, e estes apresentaram fenótipo de ressecamento e deformidades, acarretando baixa eclosão, quando comparados com o controle. Conclusões: Concluímos que o dsRNACHS administrado afeta a produção e a qualidade dos ovos das fêmeas de Ae. aegypti, sendo uma boa estratégia de controle para este inseto.

Using nod genes control system to create rhizospheric microorganisms with regulated gene expression

Inducible vector containing the full-sized nodD gene and the promoter region of the nod-box under the control of which was cloned the gfp gene was constructed. Modified bacteria R. galegae in which the synthesis of GFP protein was activated by plant flavonoids were obtained.

Matching of the GFP Gene Expression Levels by Different Terminator Sequences Regulation

The ability to express foreign genes in plant cells provides a powerful tool for studying the function of specific genes. In addition, the creation of genetically modified plants may provide new important features that are useful for industrial production or pharmaceutical applications. One of the key parameters for the development of a high level of heterologous genes expression is the efficiency of terminators used in genetic engineering, since the level of gene expression depends on its choice. Aim. Study of the gfp gene expression regulation in Nicotiana rustica L. tissues by different terminators. Methods. The Golden Gate method of molecular cloning was used for genetic constructs creation. The tissues of N. rustica plants were infiltrated by the created genetic vectors for transient gene expression. The expression level was determined by spectrofluorometric (level of green fluorescent protein (GFP) fluorescence) and protein analysis: determination of water-soluble proteins concentration and its electrophoresis separation in polyacrylamide gel (PAGE). Results. Five different terminators with polyadenylation signal/3’-untranslated region (3’UTR) were selected for the study: the 7th gene isolated from Agrobacterium tumefaciens L. (Atug7), the terminator of the gene that encode mannopinsyntase from A. tumefaciens (mas), the terminator of tomato (Solanum lycopersum L.) adenosine 5’-triphosphatase (ATPase), the potato histone H4 terminator (Solanum tuberosum L.) and the 35S Cauliflower Mosaic Virus (35S CaMV) terminator. All transcriptional units additionally contained a 5’-untranslated region out of the 2B gene from the family of genes encoding the small subunit of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (5’UTR RbcS2B), the coding sequence of the gfp gene and double 35S Cauliflower Mosaic Virus promoter (D35S CaMV). Thus, we created 5 genetic constructs with different terminator sequences. The presence of recombinant GFP protein in total protein extracts and its identity to standard protein was proved by the spectrofluorometric and PAGE analyzes. For the first time was shown the difference of GFP reporter protein accumulation in N. rustica tissues by terminator regulation of transient gfp gene expression. Conclusions. We detected the highest expression of the gfp gene when the Atug7 terminator was used and the lowest level with the histone H4 terminator. The difference between protein accumulations using these terminators was in 2.89 times. It showed that the terminator sequence has a high influence on the gene expression. It choice is an important step in genetic constructs creation, since terminator can be used for regulating the level of gene expression depending on the goals.

The influence of posttranscription silensing protein-suppressor P19 on the transient gfp gene expression level in aztec tobacco plants (Nicotiana rustica L.)

Aim. Genetic constructs creation for studying the influence effect of the viral posttranscriptional silencing protein suppressor p19 on transient reporter green fluorescent protein (GFP) expression and accumulation. Methods. The Golden Gate molecular cloning method was used to create the genetic constructs; the leafy tissues of the Aztec tobacco plants (Nicotiana rustica L.) were infiltrated with a suspension of Agrobacterium tumefaciens L.; the gfp gene expression level was determined by spectrofluorometric and quantitative protein (Bradford method) assays. Results. As a result of the work, the pSPV2324 genetic construct was created, which contained the reporter gene for the green fluorescent protein gfp and the gene for the synthesis of the viral posttranscriptional silencing protein suppressor p19 and its effect on the accumulation of the recombinant GFP protein was determined. A comparative analysis of the gfp gene expression level without and with the suppressor protein synthesis gene in the genetic vector showed that the fluorescence level of GFP protein in Aztec tobacco tissues was 1.3 times higher during spectrofluorimetric analysis using the p19 suppressor gene construct. Conclusions. The positive effect of the viral suppressor silencing P19 gene on the accumulation of recombinant GFP protein in tissues plants of N. rustica L. was shown for the first time. The increase in GFP protein fluorescence when using the p19 suppressor protein construct in spectrofluorimetric analysis coincides with an increase in the total concentration of total water-soluble proteins and the level fluorescence of GFP protein in their native electrophoretic separation. Keywords: cloning, genetic constructs, transient expression, silencing protein suppressor p19, green fluorescent protein (GFP).

A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene

Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.