[Cloning and expression of vascular endothelial growth factor receptor-1 in Escherichia coli and analysis of its growth inhibition effect on human umbilical vein endothelial cells]

Angiogenesis a process that results in neo-vascularization is an essential stage in growth of solid tumors and the formation of metastases. Vascular endothelial growth factor [VEGF] and its receptors, VEGFR1 [Flt-1] and VEGFR2 [KDR/Flk-1], are the major regulators for tumor angiogenesis. Recent studies showed that second domain of VEGFR-1is a key factor for VEGF/VEGFR-1 interaction. In this study, after RNA purification and cDNA synthesis, the second domain of VEGFR-1 [VEGFR-1-II] was amplified by PCR and cloned in T/A cloning vector. In order to increase the expression of the protein, we sub-cloned the gene into pET22b[+] and transformed the construct in Rosseta-gami 2, an efficient host for expression. The expression was induced by IPTG and confirmed by SDS-PAGE and Western Blotting. The recombinant protein was purified by IMAC column and the growth inhibition of human umbilical vein endothelial cells [HUVEC] was analyzed by the recombinant protein. The results of SDS-PAGE and Blotting confirmed the protein purification accuracy. The recombinant protein concentration was determined by Bradford protocol. The results showed that nearly 300 micri g/L VEGFR-1-II protein was produced. The function of this protein was confirmed by inhibition of HUVEC cells growth. Since this protein inhibited the angiogenesis in vitro it may be consider as an efficient anti-angiogenesis factor

efficiency of CD40 down regulation by siRNA and antisense ODN: comparison of lipofectamine and FuGENE6

Dendritic cells [DCs] are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs [siRNAs] and antisense oligodeoxynucleotides [ODN]s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs

Designing of relative quantitative Real-Time PCR for detection of CD40 gene expression in dendritic cell after suppression with antisense

Dendritic cells have a critical role in control and regulation of immune responses. It is believed that these cells can be used for the treatment of many diseases. One of the methods used in immunotherapy is based on generating of tolerogenic dendritic cells through inhibition of expression costimulatory molecules. CD40 is one of the costimulatory molecules, and inhibition of expression by antisense or siRNA techniques, can generate tolerogenic dendritic cells. Generation of tolerogenic dendritic cells will be useful in the treatment of many diseases. By developing a quantitive RT-PCR for evaluation of gene expression, generation of these cells could be possible. Using proper software we designed an Antisense and transfection of dendritic cells by lipofectamine 2000 [Invitrogen] could lead us to generate tolerogenic dendritic cells. In this study dendritic cells were extracted from of Balb/c mice Spleen and the purity of this extraction was determined by flow cytometry. BCL 1 cell line as a CD40 expressing control group and Wehi-164 cell line were cultured in RPMI-1640+10%FCS. Primer design for CD40 gene and house keeping gene [GADPH] was done by bioinformatic soft wares such as Beacon designer, mfold and Blast. RNasy plus mini kit [Qiagen] was used for RNA extraction and the Purity and Integrity were determined by O.D at 260/280 and agarose gel electrophoresis. In the next step cDNA synthesized and quantitative RT-PCR for CD 40 using IQ sybergreen [Biorad] were setup. Finally, standard curve for CD40 and internal control in different RNA concentrations were performed. After transfection with lipofectamin 2000 the amount of gene suppression were quantified by qualitative RT-PCR. Using gradient real time PCR, optimum annealing temperature, C[t] and delta Rn for CD40 and GADPH were determined, annealing temperature was 59.5 degree sign c and melting temperature was 84 degree sign c. Slope of the curve and the efficacy of PCR for CD40 and GADPH genes were quantified by serial dilution method. CD 40 Standard curve efficiency is 96.5 and the slope is -3.408 and the efficacy of GADPH standard curve is 94.1 and its slope is -3.471. The amount of CD40 gene suppression by antisense in dendritic cells was 1/32 and in BCL1 cell line was 1/64. Also the transfection reagent had no effect on the gene expression. The best time for CD40 gene expression is 48h after transfection. Semi quantitative PCR, absolute quantitative PCR and relative quantitative PCR are used to evaluative the expression of different genes such as CD40. In this study we found that for making CD40 and GADPH standard curves, Biorad two steps PCR kit [IQ -sybergreen] and Oligo dt for cDNA synthesis were very suitable. In this case, GADPH is a good internal control. Also for evaluation of gene suppression relative quantitative RT-PCR was more efficient compare to other methods such as northern blotting. The CD40 gene Suppression after 48 hours in dendritic cells was 1/32 and in BCL1 cells was 1/64