Mild and transient heat shock enhances DNA integration following lipofection of recombinant plasmids in 4T1 cells

316-320Cancer cells having stably integrated genes encoding tumor-associated antigens could be utilized as a vaccine, in-vitro stimulators of antigen-primed T-cells, and target for cytotoxicity assay, etc. Lipofection is a simple and safer technique for stable transfection of plasmid DNA. However, the poor rate of genomic integration has limited its application. In the current study, the effect of mild and transient heat shock following lipofection on the improvement of genomic integration was evaluated. The cDNA fragments encoding chicken MMP-11peptide (V32-K365) and the immunoglobulin-like domain 2 of chicken VEGFR-2 were cloned separately into pcDNA3.1 vector. Lipofection was carried out using Lipofectamine® 2000 (Life Technologies, USA) in 4T1 cells followed by a heat shock at 42°C for 10 min. Transfected cells were selected for a period of four weeks against 500 µg/mL G418 in RPMI 1640 media supplemented with 10% fetal bovine serum. Distinct G418-resistant colonies appeared after 14 days of selection. Heat shock significantly (P <0.05) increased the number of viable colonies following antibiotic selection. The immunofluorescent study confirmed the stable integration of the target DNAs into the cells. It is concluded that mild and brief heat shock following lipofection improves the stable integration of recombinant pcDNA3.1 plasmids into 4T1 cells

Mild and transient heat shock enhances DNA integration following lipofection of recombinant plasmids in 4T1 cells

Cancer cells having stably integrated genes encoding tumor-associated antigens could be utilized as a vaccine, in-vitro stimulators of antigen-primed T-cells, and target for cytotoxicity assay, etc. Lipofection is a simple and safer technique for stable transfection of plasmid DNA. However, the poor rate of genomic integration has limited its application. In the current study, the effect of mild and transient heat shock following lipofection on the improvement of genomic integration was evaluated. The cDNA fragments encoding chicken MMP-11peptide (V32-K365) and the immunoglobulin-like domain 2 of chicken VEGFR-2 were cloned separately into pcDNA3.1 vector. Lipofection was carried out using Lipofectamine® 2000 (Life Technologies, USA) in 4T1 cells followed by a heat shock at 42°C for 10 min. Transfected cells were selected for a period of four weeks against 500 µg/mL G418 in RPMI 1640 media supplemented with 10% fetal bovine serum. Distinct G418-resistant colonies appeared after 14 days of selection. Heat shock significantly (P &lt;0.05) increased the number of viable colonies following antibiotic selection. The immunofluorescent study confirmed the stable integration of the target DNAs into the cells. It is concluded that mild and brief heat shock following lipofection improves the stable integration of recombinant pcDNA3.1 plasmids into 4T1 cells

Comparative phytochemical analysis and antioxidant activity of triphala Mashi and triphala

No Abstract.Nigerian Journal of Natural Products and Medicine Vol. 10 () 2006: pp.73-7

Inhibitory Receptors Induced by VSV Viroimmunotherapy Are Not Necessarily Targets for Improving Treatment Efficacy

Systemic viroimmunotherapy activates endogenous innate and adaptive immune responses against both viral and tumor antigens. We have shown that therapy with vesicular stomatitis virus (VSV) engineered to express a tumor-associated antigen activates antigen-specific adoptively transferred T cells (adoptive cell therapy, ACT) in vivo to generate effective therapy. The overall goal of this study was to phenotypically characterize the immune response to VSV+ACT therapy and use the information gained to rationally improve combination therapy. We observed rapid expansion of blood CD8+ effector cells acutely following VSV therapy with markedly high expression of the immune checkpoint molecules PD-1 and TIM-3. Using these data, we tested a treatment schedule incorporating mAb immune checkpoint inhibitors with VSV+ACT treatment. Unlike clinical scenarios, we delivered therapy at early time points following tumor establishment and treatment. Our goal was to potentiate the immune response generated by VSV therapy to achieve durable control of metastatic disease. Despite the high frequency of endogenous PD-1+ TIM-3+ CD8+ T cells following virus administration, antibody blockade did not improve survival. These findings provide highly significant information about response kinetics to viroimmunotherapy and juxtapose the clinical use of checkpoint inhibitors against chronically dysfunctional T cells and the acute T cell response to oncolytic viruses