Avicenna Journal of Medical Biotechnology
Volume:5 Issue: 2, Apr-Jun 2013

Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods.

Gp41 of HIV (Human Immunodeficiency Virus) is a protein that mediates fusion between viral and cellular membranes. The agent, T-20, which has been approved for HIV inhibition, can restrain Gp41 function in the fusion process; nevertheless, it has disadvantages like instability, high cost of production and injection form to be delivered twice a day. Several molecules like NB-2 and NB-64 have been discovered that can inhibit HIV infection. These molecules were used as template compounds to design and develop more effective small molecules functioning as HIV-1 fusion inhibitors targeting Gp41. The process included in silico docking protocols using HEX and ArgusLab applications. A multisource database was created, after choosing the best molecules; they were tested in vitro for inhibitory activity by HIV-1 single-cycle model, transfected in HEK cells (293T). Computational analysis and experimental data were combined to explore molecular properties and the most potent ones were found, with the best suitable criteria for interaction with Gp41. Several examples (DAA-6, DAA-9 and DAA-12) could inhibit infection in vitro as effective as NB-2, NB-64. Since disadvantages of available fusion inhibitor (T-20), it seems necessary to find similar molecules to be approved and have small size providing suitable bioactivity profile. The molecules explored in this study can be good candidates for further investigations to be used as oral HIV fusion inhibitors in the future.

Trastuzumab (Herceptin) is a humanized monoclonal antibody (mAb) which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method. Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l/day. According to the results, the produced mAb had similar affinity to HER2+ tumor cells to that of Herceptin. High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase (DHFR). It is usually accepted that DHFR gene can be amplified in DHFR− CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR.

Magnetic nanoparticles in a variable magnetic field are able to produce heat. This heat (42-45°C) has more selective effect on fast dividing cancer cells than normal tissues. In this work magnetite nanoparticles have been prepared via co-precipitation and phase identification was performed by powder x-ray diffraction (XRD). Magnetic parameters of the prepared nanoparticles were measured by a Vibrating Sample Magnetometer (VSM). A sensitive thermometer has been used to measure the increase of temperature in the presence of an alternating magnetic field. To evaluate the cytotoxicity of nanoparticles, the suspended magnetite nanoparticles in liquid paraffin, doxorubicin and a mixture of both were added to the MDA-MB-468 cells in separate 15 ml tubes and left either in the RT or in the magnetic field for 30 min. Cell survival was measured by trypan blue exclusion assay and flow cytometer. Particle size distribution of the nanoparticles was homogeneous with a mean particles size of 10 nm. A 15°C temperature increase was achieved in presence of an AC magnetic field after 15 min irradiation. Biological results showed that magnetite nanoparticles alone were not cytotoxic at RT, while in the alternative magnetic filed more than 50% of cells were dead. Doxorubicin alone was not cytotoxic during 30 min, but in combination with magnetite more than 80% of the cells were killed. It could be concluded that doxorubicin and magnetite nanoparticles in an AC magnetic field had combinatory effects against cells.

Despite major progress in our general knowledge related to the application of adult stem cells, finding alternative sources for bone marrow Mesenchymal Stem Cells (MSCs) has remained to be challenged. In this study successful isolation, multilineage differentiation, and proliferation potentials of sheep MSCs derived from bone marrow, adipose tissue, and liver were widely investigated. The primary cell cultures were prepared form tissue samples obtained from sheep 30-35 day fetus. Passage-3 cells were plated either at varying cell densities or different serum concentrations for a week. The Population Doubling Time (PDT), growth curves, and Colony Forming Unit (CFU) of MSCs was determined. The stemness and trilineage differentiation potential of MSCs were analyzed by using molecullar and cytochemical staining approaches. The data was analyzed through one way ANOVA using SigmaStat (ver.2). The highest PDT and lowest CFU were observed in adipose tissue group compared with other groups (p<0.001). Comparing different serum concentrations (5, 10, 15, and 20%), irrespective of cell sources, the highest proliferation rate was achieved in the presence of 20% serum (p<0.001). Additionally, there was an inverse relation between cell seeding density at culture initiation and proliferation rate, except for L-MSC at 300 cell seeding density. All three sources of fetal sheep MSCs had the identical trilineage differentiation potential. The proliferative capacity of liver and bone marrow derived MSCs were similar at different cell seeding densities except for the higher fold increase in B-MSCs at 2700 cells/cm2 density. Moreover, the adipose tissue derived MSCs had the lowest proliferative indices.

Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotype-specific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System (TTSS) of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli (E. coli), the feasibility of the expression of this protein in tobacco has been investigated. The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens (A. tumefaciens) strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum (N. tobaccum) leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody. The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable. This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting.

Detection of Circulating Fetal Trophoblastic Cells (CFTC) by single cell genotyping not only allows to identify fetal cells from maternal blood, but also to characterize their bi-parental genome. We have tested intact fetal trophoblastes recovered at 4th to 10th weeks of gestation (WG) from blood (10 ml per mother) of 13 women after In Vitro Fertilization (IVF) and transfer of one or several embryos. Large cells isolated from blood were individually microdissected and studied by genetic fingerprinting with a mean number of 3 Short Tandem Repeats (STR) markers, known to be informative by testing paternal and maternal blood DNA. CFTC were found in all mothers starting from the 5th WG. A mean number of 2.5 CFTC per ml of blood was found in all the analyzed samples collected at the different terms of pregnancy. All mothers who received the transfer of two or three embryos, including one who delivered twins and one with vanishing twin (identified by ultrasounds), were found to have CFTC with two or three different bi-parental genotypes, belonging to different embryos derived from the same parents. CFTC circulation is detectable starting from the 5th WG. A "vanishing twin" phenomenon frequently develops after IVF and transfer of multiple embryos, being undetectable by ultrasounds and revealed by genetic CFTC fingerprinting.

The basal medium that supports Isolated Mouse Oviduct (IMO) is important for supporting embryo development and quality. The culture of ovine IVM/IVF zygotes was done in IMO using SOFaaciBSA and SOFaaBSA as basal medium of IMO and in SOFaaBSA alone as control. For preparation of IMO mature inbred strain C57BL/6 female mice were synchronized and mated with vasectomized males. The females with vaginal plug were sacrificed and the zygotes were transferred in to the isolated oviduct at 20 hpi. The oviducts were cultured with SOFaaciBSA and SOFaaBSA for 6 days. Another group of zygotes were cultured in SOFaaBSA alone as control. Culture of zygotes in the IMO with SOFaaciBSA and SOFaaBSA, did not significantly affect the development and quality of embryos (p>0.05). The hatching rate, total and trophectoderm cells number in IMO groups’ blastocysts were significantly higher than SOFaaBSA alone. The morphological appearance of IMO blastocysts was superior to SOFaaBSA alone. When the quality of oocytes was poor, IMO could better support ovine embryo development either with SOFaaBSA or SOFaaciBSA than SOFaaBSA alone and there was a significant difference in blastocyst formation at day 6 with SOFaaBSA alone. The culture of ovine IVM/IVF zygotes in IMO using two highly efficient ruminant embryo culture media not only could support development of ovine embryos similar to the level in non IMO culture system (SOFaaBSA alone) but also could improve the quality of resulting embryos. Additionally, IMO could better support the development of ovine embryos derived from poor quality oocytes compared to the SOFaaBSA alone.