Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line.

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

The Combinatorial Effect of Ad-IL-24 and Ad-HSV-tk/GCV on Tumor Size, Autophagy, and UPR Mechanisms in Multiple Myeloma Mouse Model.

Multiple myeloma is a type of malignant neoplasia whose treatment has changed over the past decade. This study aimed to investigate the effects of combination of Adenovector-carrying interleukin-24 and herpes simplex virus 1 thymidine kinase/ganciclovir on tumor growth, autophagy, and unfolded protein response mechanisms in mouse model of multiple myeloma. Six groups of mice, including Ad-HSV-tk/GCV, Ad-IL-24, Ad-HSV-tk/IL-24, Ad-GFP, and positive and negative controls, were investigated, and each group was injected every 72 h. The tumor size was measured several times. The expression of LC3B evaluated through western blotting and ASK-1, CHOP, Caspase-3, and ATF-6 genes in the UPR and apoptosis pathways were also analyzed by the quantitative polymerase chain reaction (qPCR) method. The present results showed that the injection of Ad-HSV-tk/GCV, Ad-HSV-tk/IL-24, and metformin reduced the tumor size. The expression of LC3B was significantly higher in the treatment groups and positive control groups compared to the negative control group. The expression of CHOP, caspase-3, and ATF-6 genes was significantly higher in the Ad-IL-24 group compared to the other treatment groups. Besides, the ASK-1 expression was significantly lower in the Ad-IL-24 group as compared to the other groups. Overall, the results indicated that the presence of the HSV-tk gene in the adenovectors reduced the size of tumors and induced autophagy by triggering the expression of LC3B protein. The presence of the IL-24 might affect tumor growth but not as much the therapeutic effect of HSV-tk. Furthermore, the results indicated that co-administration of IL-24 and HSV-tk had no synergistic effect on tumor size control.

Omicron-specific and bivalent omicron-containing vaccine candidates elicit potent virus neutralisation in the animal model.

Omicron variant (B.1.1.529) is able to escape from naturally acquired and vaccine-induced immunity, which mandates updating the current COVID-19 vaccines. Here, we investigated and compared the neutralising antibody induction of the ancestral variant-based BIV1-CovIran vaccine, the Omicron variant-based BIV1-CovIran Plus vaccine, and the novel bivalent vaccine candidate, BBIV1-CovIran, against the Omicron and ancestral Wuhan variants on the rat model. After inactivating the viral particles, the viruses were purified and formulated. Bivalent vaccines were a composition of 2.5 µg (5 µg total) or 5 µg (10 µg total) doses of each ansectral-based and Omicron-based monovalent vaccine. Subsequently, the potency of the monovalent and bivalent vaccines was investigated using the virus neutralisation test (VNT). The group that received three doses of the Omicron-specific vaccine demonstrated neutralisation activity against the Omicron variant with a geometric mean titer of 337.8. However, three doses of the Wuhan variant-specific vaccine could neutralise the Omicron variant at a maximum of 1/32 serum dilution. The neutralisation activity of the Omicron-specific vaccine, when administered as the booster dose after two doses of the Wuhan variant-specific vaccine, was 100% against the Omicron variant and the Wuhan variant at 1/64 and 1/128 serum dilution, respectively. Three doses of 5 µg bivalent vaccine could effectively neutralise both variants at the minimum of 1/128 serum dilution. The 10 µg bivalent vaccine at three doses showed even higher neutralisation titers: the geometric mean of 388 (95% CI 242.2-621.7) against Omicron and 445.7 (95% CI 303.3-655.0) against Wuhan. It is shown that the candidate bivalent and Omicron-specific vaccines could elicit a potent immune response against both Wuhan-Hu-1 and Omicron BA.1 variants.