Background and purpose: The optimization of an effective non-viral gene delivery method for genetic manipulation of primary human T cells has been a major challenge in immunotherapy researches. Due to the poor TRANSFECTION efficiency of conventional methods in T cells, there has been an effort to increase the TRANSFECTION rate in these cells. Protamine is an FDA-approved compound with a documented safety profile that enhances DNA condensation for gene delivery. Experimental approach: In this study, the effect of protamine sulfate on the TRANSFECTION efficiency of standard TRANSFECTION reagents, was evaluated to transfect primary human T cells. In this regard, pre-condensation of DNA was applied using protamine, and the value of the zeta potential of DNA/protamine/cargo complexes was determined. T cells were transfected with DNA/protamine/cargo complexes. The TRANSFECTION efficiency rate was evaluated by flow cytometry. Also, the green fluorescent protein expression level and cytotoxicity of each complex were identified using real-time polymerase chain reaction and MTT assay, respectively. Findings/Results: Our results demonstrated that protamine efficiently increases the positive charge of DNA/cargo complex without any cytotoxic effect on the primary human T cells. We observed that the TRANSFECTION efficiency in DNA/protamine/ Lipofectamine ® 2000 and DNA/protamine/TurboFect TM was 87. 2% and 78. 9%, respectively, while TRANSFECTION of T cells by Lipofectamine ® 2000 and TurboFect TM would not result in sufficient TRANSFECTION. Conclusion and implications: Protamine sulfate enhanced the TRANSFECTION rate of T cells; and could be a promising non-viral gene delivery method to achieve a safe, rapid, cost-effective, and efficient system which will be further applied in gene therapy and T cells manipulation methods.

Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on TRANSFECTION efficiency of some commercial reagents used for TRANSFECTION of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different TRANSFECTION reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial TRANSFECTION reagents in different combinations. The TRANSFECTION permissible HEK293-FT cell line was used as a control in TRANSFECTION procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post TRANSFECTION. Our results showed TRANSFECTION efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after TRANSFECTION with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production.

Introduction: Neural stem cells (NSCs) are multipotent stem cells residing in the central nervous system that is capable of self-renewal to support ongoing requirements for neurogenesis in the adult brain. Since NSCs are considered potential candidate cells for neuro-regenerative medicine, applying safe induction methods for them is very important. Synthetic modified-mRNA (mmRNA) as an alternative to traditional DNA-or protein-based methods, is regarded as a powerful tool for inducing short-term gene expression in cells with no genetic manipulation. Methods: Here, we aimed to develop an optimized condition for mmRNA TRANSFECTION in primary NSCs. In vitro-transcribed EGFP mmRNA (mmRNAEGFP) was delivered to human embryonic kidney cells (HEK293T) and mouse NSCs by using two commercial agents, Lipofectamine-2000 (LF2000) and TransIT. Also, a plasmid DNA was used to transfect cells considered EGFP-expressing positive control. In addition, the poly(A) tail (poly adenosine tail) elongation and chloroquine (CQ) treatment were performed to improve TRANSFECTION efficiency. Finally, flow cytometry, fluorescence microscopy, and MTT assays were performed to assess the cells. Results: In comparison with HEK293T, NSCs were very sensitive to TRANSFECTION, the efficacy of TRANSFECTION using DNA/LF2000 was higher in HEK293T cells, but mmRNAEGFP/ TransIT showed better TRANSFECTION efficacy in NSCs. Poly(A) tail elongation,also, treating the cells with CQ before TRANSFECTION significantly improved its efficacy. Conclusion: The mmRNA poly(A) tail elongation and the use of specific TRANSFECTION agents in combination with TLR inhibitors can lead to a more effective TRANSFECTION in NSCs.

Objective: (i) if using chemical reagents such as DMSO and Lipofectamine™2000 increase bovine sperm TRANSFECTION, (ii) if motile spermatozoa have a greater TRANSFECTION rate than immotile ones, and (iii) if increasing sperm- DNA incubation time enhance sperm TRANSFECTION rate.Materials and Methods: Motile bull sperms were allowed to swim up at 38oC for 90 min. The rhodamine labeled pGeneGrip-Lac-Z vector (Genelantic, USA) was used for direct detection of transfected cells under epiflourescence microscope. One and three microlitersof Lipofectamine2000 were added separately into two tubes containing 20μl of SP-TALP medium (BSA and antibiotic free) and stored at room temperature for 30 min. One microgram of the vector was added to liposomes and stored at room temperature for a further 90 min to perform 1:1 and 1:3 (DNA: Lipofectamine) ratios. one mg of naked DNA was added into SP-TALP medium as control group and supplemented with 1% and 3% Dimethyl sulfoxide(DMSO) as DMSO groups. for all groups, 1×106 fully capacitated spermatozoa was added to each tube and the total volume adjusted to 30ml with SP-TALP medium (containing 6% BSA) and incubated at 38oC for 60 min. Sperm-DNA mixtures were divided into two groups, treated with or without DNaseI (5 UI) for 30 min and used for fluorescence detection. In another