Cell Journal (Yakhteh)
Year:2009 | Volume:11 | Issue:2 (42)|Papers Count:24

Over the past two decades, many approaches for transferring genes into the genome of domestic animals have been devised. The main purposes of transgenesis are: to increase animal capabilities, to knock down or silence both onco-genes and deleterious genes, and to produce a pharmaceutical protein. Transgenesis techniques include pronuclear microinjection (PNM), somatic cell nuclear transfer (SCNT), viral infection (VI) and sperm-mediated gene transfer (SMGT). The first transgenic mouse was produced by using the PNM technique and transgenic animals from other species (rabbit, sheep, pig and cattle) have been produced thereafter. However, the PMN technique had certain drawbacks: low efficiency, random integration site of the transgene and a high mosaic rate. For this reason, other alternative techniques have been devised to overcome its drawbacks. The most reliable method for transgenesis which bypasses mosaics is SCNT. However, this method is complicated and tedious; with multiple stages that need setting up. VI has been used to transfer genes into the oocytes and zygotes with high efficiency and versatility. In spite of its simplicity, the maximum transgene length should be less than 8.5 kb. Currently, spermatozoa are considered as an alternative method of carrying transgenes into the oocytes with minimum technical demands. In contrast to VI, SMGT is being used successfully to transfer different kinds of BACs with more than 200 kbp into mouse oocytes. The present review summarizes the methods by which transgenes can be introduced into zygotes of domestic animals.

Since the 1970s when long term potentiation (LTP) was introduced to the scientific world; several studies have been devoted to determining whether this phenomenon is naturally a basic mechanism of learning and memory in mammalian brains. However, plenty of evidence confirms that a) LTP is inducible in the circuits involved in learning and memory; b) common receptors and intracellular cascades are recruited in both memory and synaptic plasticity and c) LTP and memory are similarly affected by many parameters such as: ligands, environmental signals, history of neuronal activity. Despite this, contradictory reports exist which oppose the similarities between LTP and memory. In this paper we briefly introduce learning, memory and LTP, and argue relevant factors that possibly connect them. Ultimately, current considerations lead one to conclude that the time is too early to judge clearly if LTP is a real mechanism of learning and memory.

Matrix metalloproteinases are zinc and calcium dependent endopeptidase families that are expressed in injured tissue such as cardiovascular or hepatic disease. Complex efforts of this enzymes on the extra cellular matrix structure is related to up and down regulation of them and their tissue inhibitors. Configuration of extra cellular matrix during pathogenesis, curing and development is affected by two key mechanisms: matrix metalloproteinase and hepatic stellate cell activity. The important role of these enzymes on liver injuries and regeneration are indicated when their effects on migration of bone marrow stem cells and hepatic stem cells was discovered.

Objective: Polytope DNA vaccines, capable of focusing the cytotoxic T lymphocyte (CTL) response on critical epitopes, represent a promising approach in HCV immunotherapy. Nevertheless, due to controversial rules governing epitope processing and the low level expression/ immunogenicity of recombinant polytope peptides, designing and primary expression/ immunogenicity analysis of these vaccine types should be the first consideration prior to costly transgenic animal studies.Materials and Methods: Four HLA-A2 and H-2d restricted CTL epitopes were selected and designed in three appropriate sequential tandems based on epitope and proteasomal cleavage predictions. The related nucleotide sequences were synthesized using SOEing PCR method and cloned into a pcDNA3.1 vector, either alone or fused to the small hepatitis B surface antigen (HBsAg-S) gene. Following the preparation of polyclonal anti-sera, expression/ secretion of polytopes was evaluated in Cos-7 cells by using immunofluorescence, Westernblot, dot blot, ELISA and RT-PCR techniques. The immunogenicity of the plasmids was also assessed through the delayed-type hypersensitivity (DTH) assay in BALB/c mice.Results: Due to in silico designs and optimizations, the polytope products of constructed plasmids were efficiently detected in vitro through common techniques and HBsAg-S-based particles were shown to be secreted into the culture media (up to 30%). Moreover, all plasmids were able to efficiently induce a positive DTH response while HBsAg-S fusion constructs indicated a significant immunopotential effect towards the incorporated mouse epitopes.Conclusion: Designed polytope constructs of this study are efficiently expressed and processed. They have the required initial potency for further immunogenicity analysis in transgenic mice.

Objective: Freshwater planarians were used as models for studying metazoan regeneration and stem cell biology. Here a simple, fast and high throughput method for extracting their stem cells (neoblasts) is represented.Materials and Methods: Specimens of the Dugesia sp with an average length of 18mm were homogenized by a glass Dounce tissue grinder which contained about 1 ml of planarian saline solution. The extracted suspension was serially filtered by 60, 41, 30, 20 and 11mm nylon meshes. In order to obtain purified neoblasts in the final suspension; this suspension has been compared with a cell suspension from 30Gy irradiated worms. Hoechst 33342 was used to determine cells from non-cellular particles; methylene blue and propidium iodide were used to detect the number of dead cells in each extraction.Results: About 2.6-3 million cells were extracted from 10-12 worms. Flow cytometry analyses showed about 83% of the extracted particles were cells. In suspensions from irradiated animals, about 50% of the cells were absent; the final suspension contained about 62-66% neoblasts and about 17% non-cellular particles. When these extracts were treated with distilled water to destroy the cells, only rabdites and chitinous spines of the parenchyma were observed in the extract.Conclusion: Results show that the purity of neoblasts in the final suspension is about 66%. Non-cellular particles have a carbohydrate nature and, therefore, this extraction method is completely compatible with molecular (e.g. proteomics and transcriptomics) and cellular methods (e.g. neoblast culture).

Introduction: Wnt and k-ras are two main signaling pathways activated in colon cancer. Many genes are upregulated downstream of these signaling pathways. The aim of this study was to assess the activity of Wnt and k-ras in HCT116 and SW480 cell lines by making two reporters constructs using promoters downstream of these pathways (fibroblast growth factor18 [FGF18] and urokinase plasminogen activator receptor [UPAR]).Materials and Methods: UPARLacZ, FGF18LacZ, negative (pUCLacZ) and positive (CMVLacZ) control plasmids and pRc/CMV2CAT were constructed. Expressions of LacZ in both cell lines were studied by bgal staining and ELISA after normalization with CAT expression.Results: In both cell lines, FGF18LacZ transfected cells stained more than UPARLacZ transfected ones. This difference was more prominent in SW480. Both constructs have the ability of expression in both cell lines. It was also proven that FGF18LacZ was significantly more active than UPARLacZ in both cell lines. Expression of FGF18LacZ in HCT116 and SW480 cell lines was respectively 1.34 and 4.4 times more than UPARLacZ.Conclusion: Despite the fact that in HCT116 the Ras pathway is activated, FGF18LacZ is more active than UPARLacZ although the UPAR promoter is more active in HCT116 cell line than SW480 cell line. These findings are in accordance with previous studies that in all colon cancer cell lines Wnt signaling pathway is active even though there is no mutation in any part of it. Wnt is the main signaling pathway responsible for carcinogenesis in colon epithelial cells. These constructs can be used as reporters for studying the above mentioned signaling pathways in other cell lines.

Objective: Amino acid alignment analysis of deduced amino acid residues revealed a tripeptide (SKI) at the carboxy terminus of peroxisomal protein cDNA. In order to see the importance of the above sorting signal, we have performed a site-directed mutagenesis to delete SKI tripeptide and its transfection into CHO-K1 and P19 cells.Materials and Methods: In order to create the appropriate site-directed mutant, PCR was done with specific primers. Amplified PeP cDNAs either containing SKI or deleted ones were constructed downstream of EGFP cDNA under regulation of the cytomegalovirus (CMV) promoter in a pEGFP-C1 vector. Transfection of P19 and CHO cells was done with lipofectamine2000.Results: Gradient PCR showed that the best annealing temperature was 71.6oC. Transfection of plasmids containing chimera of EGFP-PEP cDNAs into the CHO-K1 and P19 cells showed several punctuate structures, presumably peroxisomes, while SKI deletion showed a cytosolic mislocalization of the EGFP pattern.Conclusion: Taken together, these data strongly suggest that SKI, which is located at the C- terminus of the protein, is required for sorting this protein.

Objective: It has been demonstrated that mesenchymal stem cells (MSCs), which are isolated from various tissues, have different potential in differentiation and proliferation. For this reason it is necessary to isolate these cells from various sources in order to use them in clinical settings. The present study has been done to investigate the possibility of isolation, culture and characterization of human synovium-derived mesenchymal stem cells.Materials and Methods: Samples (200-300mg) were provided from synovium tissue of patients who had knee surgery. Obtained samples were homogenized, enzymatically minced with collagenase D and passed through 70mm nylon filters; separated cells were then cultured. Isolated cells were identified by morphological observations, differentiation tests, flow cytometry, immunocytochemistery studies and RT-PCR.Results: The isolated cells in this study showed fibroblast-like morphology and have a high proliferation capacity. In flow cytometry and immunocytochemical studies, they were positive for CD73 and CD105 antigens. RT-PCR analysis and specific staining for differentiated cells towards osteogenic and adipogenic lineages, showed that isolated cells were potent in differentiation into the mentioned lineages.Conclusion: These results suggest that synovium tissue, which is discarded in most knee operations, can be used for cell therapy and tissue engineering protocols as an enrichment source of potent mesenchymal stem cells.

Objective: To examine the efficiency of both the Zeta and density gradient centrifugation (DGC) methods for the selection of normal chromatin sperm by TUNEL, sperm chromatin dispersion (SCD), acridine orange and chromomycin A3 (CMA3).Materials and Methods: The study was performed on 63 patients. Semen analysis was carried out according to WHO criteria. Semen samples were divided into three equal portions. One portion was used as the control, the second portion was used for the Zeta method and the third portion underwent DGC method. All portions were evaluated for sperm morphology (Diff Quick staining), protamine deficiency (CMA3) and DNA integrity (SCD, AO and TUNEL). Coefficients of correlation and student’s t test were carried out using SPSS 11.5.Results: The mean percentage of abnormal sperm, protamine deficiency and DNA fragmentation in the Zeta and DGC methods were significantly decreased as compared to the control group (p<0.005). In addition, the DGC method was higher in comparison to the Zeta method in the selection of sperm with normal morphology (p<0.005). The Zeta method was higher in comparison to the DGC method in the selection of sperm with intact DNA (p<0.005).Conclusion: Both Zeta and DGC methods were effective in the selection of sperm with better quality in terms of normal morphology, normal protamine content and DNA integrity. Therefore, we suggested that the combined Zeta and DGC method should be used for selection of normal sperm.

Objective: The aim of this study was to investigate the effects of a 217 Hz magnetic field of mobile phone GSM 900 exposure on the bioelectric activity of F1 neuronal cells of the land snail.Materials and Methods: According to the magnetic field measurement of the mobile phone, a range of flux intensities of magnetic fields (0.46 - 229mT) at a frequency of 217 Hz was produced by magnetic field coils. The bioelectrical activity of F1 nerve cells at different time intervals was recorded, using intracellular recording under current clamp conditions in control, sham and field exposed groups.Results: Magnetic field exposure decreased the amplitude of action potential and the firing frequency of F1 nerve cells. Furthermore, it resulted in a significant (p<0.05) increase in the amplitude of after hyperpolarization (AHP) and duration of action potential. Change in the cell’s electrophysiological parameters was associated with a decrease in neuronal excitability. Magnetic field exposure affected also the resting membrane potential of F1 cells in a bimodal fashion, including depolarization and hyperpolarization. Considering the exposure condition, most of the alterations in the electrical activity of F1 nerve cells induced by magnetic fields exposure were reversible.Conclusion: These findings suggest that 217 Hz magnetic fields of mobile phones with different intensities affect the spontaneous bioelectrical activity of F1 nerve cells and exert inhibitory effects on neuronal excitability. There is evidence for the existence of an amplitude window and these electrophysiological alterations occur within this amplitude window. The reversibility of the magnetic field- induced most electrophysiological alterations in the neuronal behavior under our experimental conditions was observed.

Objective: Anti-cancer therapies frequently lead to ovarian damage and defective fertility. To preserve fertility, cryopreservation and subsequent transplantation of the ovaries has been suggested. The aim of this study was to investigate the survival of follicles in intact intramuscular mouse autologous ovaries, according to the time the ovarian tissue remained in the grafted site.Materials and Methods: Ovaries (n=9) were transplanted intramuscularly into gluteus superficialis. These grafted ovaries were removed after one, two and three weeks from the grafted site. A histological examination and counting of follicles was then performed. Some ovaries (n= 3) from the same mice were selected randomly for the control group. Hematoxyline and eosin (H & E) staining was used for follicle counting and TUNEL staining for the examination of apoptosis in grafted tissues.Results: Mean follicular survival was significantly lower in experimental groups compared to the control group (non-grafted) (p<0.05), because of ischemic damages. Also healthy primordial follicle numbers in grafted ovaries were higher than other types.Conclusion: Antral follicles are the most sensitive to ischemic damages and primordial follicles are the least sensitive. Also the presence of healthy follicles in grafted tissues shows that ovarian transplantation could be a promising method for infertility treatment of patients diagnosed with cancer.

Objective: Angiogenesis is a key process in the promotion of cancer and its metastasis. Many natural health products inhibit angiogenesis. Because of the lack of molecular studies on anti-tumor and anti-angiogenic effects of shallot (Allium ascalonicum); except a few clinical studies on other shallot properties, such as the anti-proliferative effect of shallot chloroformic extract on two tumor cell lines, the present study focuses on the anti-angiogenic effect of aqueous shallot extract using an aorta ring model.Materials and Methods: Aortic rings were obtained by cross-sectioning, at 1-2 mm intervals, the thoracic aorta of 4-8 weeks old Wistar male rats and cultured them in a thin drop of type I collagen gel. After 3 days of culturing and first sprouting, the extract of A. ascalonicum (from 25 to 800mg/ml) was added to cultures. The results of anti-angiogenic activity were investigated by microscope. The cytotoxicity of extract at different doses on HUVECs was measured by trypan blue assay.Results: The results showed that the shallot extract has suitable anti-angiogenic effect in a range of 50 to 800mg/ml, but in 25mg/ml, the extract has no considerable effect. In addition, a tangible cytotoxic effect on endothelial cells at the above mentioned doses was observed.Conclusion: Our study showed that aqueous extract of A. ascalonicum bulbs has noticeable anti-angiogenic activity without toxic effect on the cells in doses that ranged from 50-800mg/ml. Therefore, A. ascalonicum can be a potential candidate for further investigations used in angiogenesis-related pathologic conditions.

Objective: The aim of this study was to clone peroxisomal protein (PEP) cDNA in a mammalian expression vector in a chimeric cDNA type, with enhanced green fluorescent protein (EGFP) cDNA. To investigate the intracellular localization of PEP protein linked to EGFP marker, the constructed plasmid was used for transfection into the chinese hamster ovary (CHO) cells.Materials and Methods: Total RNA was extracted from the heart tissue of an adult mouse. PEP cDNA was constructed using reverse transcriptase and was amplified with specific primers covering the entire length of ORF. RT-PCR products containing PEP cDNA were treated by enzymatic digestion and inserted into the pEGFP-C1 downstream of EGFP cDNA and were used for transformation into bacterial competent cells. The positive colonies which showed inserted PEP cDNA were selected for plasmid preparations and additional analysis was performed to ensure that PEP cDNA was inserted properly. Finally, to confirm the intracellular localization of EGFP-PEP, CHO cells were transfected with the constructed plasmid.Results: Our results confirmed amplification and cloning of the expected product. PEP cDNA encompasses 630bp which encodes 209 amino acid residues. Bioinformatics analyses have shown the presence of a fibronectin type III domain (31-114a.a.) and two hydrophobic domains (12-32a.a. and 152-169a.a., respectively). Because of the presence of serine, Lysine, leucine (SKI) in the C-terminal of the related protein, transfection data showed peroxisomal localization of PEP as was similar to the catalase.Conclusion: Taken together these data showed that PEP is a peroxisomal protein. However the importance of its fibronectin type III and two hydrophobic domains should be assessed by further experiments.

Objective: It has been seen that wild waterfowls stop breeding during captivity. In the longterm, this may put their species in danger and there would be a need to find a way for artificial reproduction. In this study, a common medication for human controlled ovarian hyperstimulation (COH) was tested on wild waterfowls to answer the question of whether this application can cause ovarian follicular recruitment and does it help the fowl ovulate and lay eggs.Materials and Methods: The animal experimental model was the adult female Mallard. The timing of research was scheduled for mid-July through mid-August which counts as outofseason for Mallard breeding. 75 IU/bird/day was injected IM for 10 days. After completion of injections, the ovarian tissues were retrieved and considered for morphological and histological assessments.Results: The results show a positive effect for human menopausal gonadotropin (hMG) on most of the evaluated parameters. In the experimental group; ovarian size, number of differentiating oocytes (vitellogenic and post-vitellogenic) and theca layer diameter were significantly more than the control group (p<0.05). Differences in the other parameters (the number of undifferentiated and pre-vitellogenic oocytes, nucleus and arteriole diameter) compared between control and experimental groups were not statistically significant.Conclusion: It seems that hMG has a positive and meaningful effect on ovarian follicular recruitment and its administration will be an effective method for ovulation induction in female Mallards. This may especially be combined with artificial insemination to help the laying eggs become fertilized.

Objective: We examine the effects of serum starvation, culture to confluence, and full confluence on cell cycle characteristics and apoptosis of goat dermal fibroblast cells.Materials and Methods: Fibroblast cells were obtained from the ear of a female goat, 1.5 years of age. The following experimental groups were analyzed for fibroblast cells: 1. Cells confluent for 72h, 2. cells starved for 48 and 72h.Results: Analysis of cell cycle distribution by flow cytometry showed that 4.56, 51.88 of normal cycling cells were at the G0, G1 phases, respectively. Serum starvation for 48 and 72h arrested cells at the G0/G1 phase (p<0.05). 91.53% of full confluent cells were at G0/ G1 stage, but in contrast to the serum starved group, this high percentage of G0/G1 cells was mainly associated with G1 cells. When DNA synthesis was detected by BrdU incorporation 19.80% of normally growing cells were in S phase. The percentage of cells in S phase changed significantly among 48 and 72h serum strarvation and full confluent groups. Under normal culture conditions, 6.67% of cells underwent apoptosis. Serum starvation for 48 and 72 hours caused early apoptosis in 8.91 and 39.83 of cells, respectively. Treatment with full confluence for 72 hours did not increase the number of apoptotic cell significantly (6.39%). Serum starvation for 72 hours increased early apoptosis significantly (p<0.05).Conclusion: Goat dermal fibroblasts at various stages of the cell cycle were effectively synchronized; that could be beneficial for somatic cell nuclear transfer in this species. Although serum starvation for 72 hours effectively arrested cells at the G0/G1 stages, it significantly increased cell apoptosis.

Objective: This study introduced a simple method for bovine blastocyst vitrification.Materials and Methods: Bovine blastocysts were produced in vitro by means of a whole co-culture system with vero cells. The blastocysts were randomly divided 1:3 into either vitrification (100 blastocysts) or control (43 blastocysts) groups. For vitrification,expanded – blastocysts were incubated first in equilibration medium for 8 minutes and then in the vitrification solution for 1 minute. The blastocysts were then loaded in the tip of a handmade cryotip for immediate - deep freezing in liquid nitrogen. Frozen embryos were then warmed by directly immersing the tips in sequential warming solutions. Warmed embryos were cultured for a further period of 48 hours when the ratios of re-expansion, hatching and degeneration were compared with the control group.Results: After warming, in the vitrified and control groups the ratios of re-expansion were 78.5% ± 0.067 and 81.6% ± 0.072, the ratios of hatching were 43.7% ± 0.083 and 49.8% ± 0.089 and the ratios of degeneration were 36% ± 0.082 and 22.3% ± 0.087, respectively, which were not significantly different between the two groups.Conclusion: Post - warming survival of the vitrified and non - vitrified embryos were not significantly different, handmade cryotips can be used as an efficient and feasible device for bovine blastocysts vitrification.

Objective: Dehydroepiandroesteron (DHEA) is a neurosteroid with potential effect on neurogenesis, neuronal survival and proliferation of neural progenitor cells. However there is no direct evidence for its biological effect during the differentiation of stem cell-derived neurons. The p19 line of embryonal carcinoma cells develops into neurons, astroglia and fibroblasts after exposure to retinoic acid (RA). This study was initiated to assess the effect of DHEA on neural cells derived from p19 embryonal carcinoma stem cells.Materials and Methods: P19 cells were suspended in dulbecco’s modified eagle’s medium (DMED) containing fetal bovine serum (FBS) in bacterial-grade petri dishes in the presence of RA, DHEA and RA+DHEA for 6 days. Then cells were trypsinized for dispersion and replaced in poly L- lysine (10mg/ml) coated tissue culture dishes without RA and DHEA for 4 days. The expression of neural markers Map-2, Tau, beta-tubulin III- clone Juj (Tuj1), astrocyte marker GFAP and the percent of neurotransmitters tyrosin hydroxylase, glutamate, serotonin and actyl cholin transferase were evaluated by flowcytometry, immunocytochemistry and RT-PCR analysis.Results: Flowcytometry analysis showed that about 63 ± 3% of the cells express neuronal marker Tuj1 and about 5 ± 1% of the cells express tyrosine hydroxylase neurotransmitters in RA treated groups. However when RA and DHEA were added to the culture medium, Tuj1 expression increased to about 74 ± 1% and tyrosine hydroxylase expression increased to 23 ± 2%.Conclusion: Results showed that DHEA accompanied RA increased the number of Tuj1 and dopaminergic neurons that were derived from p19 embryonal carcinoma stem cells.

Objective: Detection of central nervous system (CNS) molecular defects in an animal model of multiple sclerosis.Materials and Methods: Experimental autoimmune encephalomyelitis (EAE) was induced by a myelin oligodendrocyte glycoprotein. Protein expression profiles in the central nervous system between healthy clinical scores 1 and 3 of EAE were studied using a two dimensional electrophoresis based proteomics approach coupled with MALDI TOF/TOF mass spectrometry.Results: We identified 8 mitochondrial proteins that were differentially expressed in CNS, all of them down-regulated in scores 1 and/or 3. Of these, 5 proteins belong to the mitochondrial respiratory chain including: NADH dehydrogenase (ubiquinone) Fe-S protein 8, cytochrome c oxidase Va, cytochrome c oxidase Vb, ATP5B, NADH dehydrogenase (ubiquinone) flavoprotein 2. We also observed down-regulation of three other mitochondrial proteins including: glutaredoxin 5, estradiol 17 beta-dehydrogenase 8 and isocitrate dehydrogenase.Conclusion: Down-regulation of mitochondrial proteins supported the hypothesis that hypoxia-like tissue injury in multiple sclerosis (MS) lesions may be due to mitochondrial impairment.

Malassezia species considered to be the etiological agents of pityriasis versicolor and Malassezia follicolitis in humans. Recently, on the basis of molecular data, four new species were added to the genus. In total, 11 species have been described and accepted so far. In this study we describe a new isolate of Malassezia based on the nucleotide sequence of 26SrDNA and ITS1 regions, as the accepted critical markers for description of the species. The yeast was isolated from a hamster. Two primer pairs, one for amplification of D1/D2- 26Sr DNA and another for the ITS1 region were used in PCR. The PCR products were sequenced and analyzed to compare with other similar sequences which are already deposited in the GenBank. The 26SrDNA PCR product was also digested with the restriction enzyme CfoI. Malassezia-specific universal primer pairs successfully amplified the 26srDNA and ITS1 regions of the new isolate, providing a single PCR product of about 580 and 280 base pairs, respectively. After digestion of the 26s PCR product with the enzyme CfoI, a unique and different RFLP pattern was observed. Sequence analysis of D1/D226s and ITS1 regions were compared with the same regions in all already described Malassezia species, which implied different and unique new sequences. The phylogenetic tree of both regions showed that the isolate could be a different Malassezia isolate. Regarding the new RFLP pattern of D1/D226SrDBA and the unique nucleotide sequence of both D1/D2 26SrDNA and ITS1 regions, we propose the isolate to be a new Malassezia.