Introduction: While human endothelial progenitor cells (EPCs) have been a subject of somehow extensive investigation, EPCs from adult mouse hematopoietic system were poorly studied. Present investigation is focused on FVB mouse endothelial progenitor cells in terms of their isolation, purification, and expansion. Materials and Methods: Mononuclear cells collected from murine peripheral blood were cultured in fibronectin coated plate for two weeks, at which point, the adherent cell population were lifted and analyzed in terms of some surface markers. Using FACS Vantage equipped with one-cell deposition unit, single CD34 positive cells were plated per well already containing medium optimized for single cell growth. Several clones were then emerged, expanded, and examined in terms of some surface markers. Furthermore, the cells were investigated regarding ability to uptake DiI-ac-LDL and form capillary network on matrigel surfaces. Results: Adherent population of mononuclear cells from mouse peripheral blood was appeared morphologically heterogeneous. About 5% of the adherent cells were CD34 positive. Having optimized their culture condition, several CD34 positive clones were expanded. The cells comprising the clones were DiI-ac-LDL+ and formed capillary-like tube when being seeded on matrigel surfaces. Conclusion: The primary culture of the mononuclear cells from murine peripheral blood contains a very limited number of cells positive for endothelial lineage markers. These cells (adherent CD34 positive) could be expanded by single cell cloning technique.

Aim and Background: Using sensitive and specific laboratory techniques such as PCR is necessary on diagnosis of hepatitis which caused by hepatitis B virus (HBV). Common serological methods didn’t have the capability of quick and accurate detection of infection detection thought molecular methods such as PCR are effective tools for evaluation prevalence of HBV. A different result for the reason of not being standard is one of the disadvantages of this rich molecular technique. One of the most significant preventives in widespread usage of diagnostic PCR is not having proper PCR internal controls. The aim of this study is designing and constructing plasmid internal control (IAC) for identification of PCR inhibitors and afterward usage in diagnostic laboratories.Materials and methods: In this study for constructing the internal control, PCR specific primers for HBsAg gene of hepatitis B virus were optimized then sensitivity and specificity were determined. Also Composite Primers for HBV-IAC were designed and amplified by PCR then cloned. Minimum number of IC in each PCR reaction was examined through dilution and PCR results with IC.Results: By using special primers HBV PCR product was 262bp and IAC-HBV product was 660bp which had desirable different in sizes. Minimum number of IC in each reaction was 1000. Maximum of PCR sensitivity with IC for hepatitis B virus DNA was determined as 16 million Particles. In specificity test with different factors no non specific product was observed.Conclusion: For Molecular detection of HBV using an internal control can recognize the errors and will standardize this sensitive technique.

How can we clone PCR products.pGEMâ‐T Easy Vector System is a convenient system for the cloning of PCR products.How is the vector prepared?The vector is prepared by cutting the pGEMâ‐T Easy Vector with EcoRV and adding a 3' terminal thymidine to both ends (Fig. I). These single 3' T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmids by preventing recircularization of the vector and providing a compatible overhang for PCR products generated by Taq as a thermostable polymerase. Taq polymerase often adds a single deoxyadenosine, in a template‐independent fashion to the 3' ends of the significant proportion of amplified fragments as shown in the Figure. Using this method, only one insert will be ligated into the vector as opposed to multiple insertions that can occur with blunt ended cloning. In addition, with T vector cloning there is no need to dephosphorylate the vector, and there is a low background of relegated vector.

Introduction: Different data resulting from non-standard molecular techniques in laboratories were considered as one of the deficiencies about amplification techniques. One of the most important aspects about the articles published in PCR detection until now, is absence of internal control (IC) in majority of them. The aim of this study is to design and develop a simple and rapid method to produce internal control for PCR test and its future application in the routine recognition laboratories.Materials and methods: To produce competitive internal control in this study, composite primers and PCR-Cloning method were used. We designed and synthesized the forward and reverse primers of PCR diagnostic test of Hepatitis B virus(HBV), Herpes simplex virus(HSV), Mycobacterium tuberculosis(MTB), Mycoplasma pneumonia(Mpn), Cryptococcus neoformans(Cne), Salmonella spp (Sal.sp) and Mycoplasma spp (M.sp) in the 5’ primers of Leishmania Kintoplast gene in the tail form. The amplified internal controls were attached to pTZ57R and transformed in E.coli JM107 bacteria. The minimum IC number was studied using dilution technique and also PCR response spectrum with IC.Results: The size of HBV, HSV, MTB, MPN, C.ne, Sal.sp, M.sp diagnostic product with specific primers were 262, 454, 245, 345, 415, 284, 272 bp and corresponding internal control also were 660, 662, 660, 669, 661, 668, 672 bp respectively, and desirable difference observed between PCR, IC regarding the size was easy to separate in the gel. The minimum IC was identified to 1000 in each reaction and maximum PCR test sensitivity with IC was provided for entire agents appropriately. Further, in specific test using different agents any unwanted product was not observed too.Discussion and conclusion: The negative and positive false results in PCR tests are one of the difficulties of this exacting technique which may lead to decrease its performance. Using an internal control in molecular detection method as an internal controlling system, could identify these errors.

Background: With respect to the prevalence of leishmaniosis in our country and the problems occurred in the therapeutic approaches and also with respect to the previous successful reports of paromomycin efficacy in this regard, the present study was conducted to determine the effects of paromomycin and gentamicine sulphate on promastigotes of Leishmania major. Materials and methods: It was an experimental study. The base of films composed of ethyl cellulose and HPMC (hydroxyl propyl methyl cellulose) containing paromomycin 15% and gentamicine 0.5%. To determine the rate and duration release from the films and its killing effects on leishmania promastigotes, a cloning system of parasite was established by using a set of modified NNN medium without liquid layer. The medium plates were divided in 3 groups, the media contain of drug films, placebo films, and media containing no film. Results: results showed no colonies of Leishmania in a 30mm radius envelop the drug films. On the other hand, grown colonies of Leishmania promastigotes were found in placebo and control plates. Frequency of dead parasites were 98% and 5% in the placebo-control plates and drug plates, respectively (p<0.000). Conclusion: Paromomycin and gentamicine drug films have good potential of drug releasing and suitable killing effects in vitro condition. It is suggested that the therapeutic effect of this new form of drug be achieved in human models.