Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C

BACKGROUND: Splicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches. RESULTS: A method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C) incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product. CONCLUSION: We have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers

Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo

A comparison of polarized and non-polarized human endometrial monolayer culture systems on murine embryo development

BACKGROUND: Co-culture of embryos with various somatic cells has been suggested as a promising approach to improve embryo development. Despite numerous reports regarding the beneficial effects of epithelial cells from the female genital tract on embryo development in a co-culture system, little is known about the effect of these cells when being cultured under a polarized condition on embryo growth. Our study evaluated the effects of in vitro polarized cells on pre-embryo development. METHODS: Human endometrial tissue was obtained from uterine specimens excised at total hysterectomy performed for benign indications. Epithelial cells were promptly isolated and cultured either on extra-cellular matrix gel (ECM-Gel) coated millipore filter inserts (polarized) or plastic surfaces (non-polarized). The epithelial nature of the cells cultured on plastic was confirmed through immunohistochemistry, and polarization of cells cultured on ECM-Gel was evaluated by transmission electron microscopy (TEM). One or two-cell stage embryos of a superovulated NMRI mouse were then flushed and placed in culture with either polarized or non-polarized cells and medium alone. Development rates were determined for all embryos daily and statistically compared. At the end of the cultivation period, trophectoderm (TE) and inner cell mass (ICM) of expanded blastocysts from each group were examined microscopically. RESULTS: Endometrial epithelial cells cultured on ECM-Gel had a highly polarized columnar shape as opposed to the flattened shape of the cells cultured on a plastic surface. The two-cell embryos cultured on a polarized monolayer had a higher developmental rate than those from the non-polarized cells. There was no statistically significant difference; still, the blastocysts from the polarized monolayer, in comparison with the non-polarized group, had a significantly higher mean cell number. The development of one-cell embryos in the polarized and non-polarized groups showed no statistically significant difference. CONCLUSION: Polarized cells could improve in vitro embryo development from the two-cell stage more in terms of quality (increasing blastocyst cellularity) than in terms of developmental rate

Studies of melatonin effects on epithelia using the human embryonic kidney-293 (HEK-293) cell line

The expression of melatonin receptors (MR) of the Mel(1a) subtype in basolateral membrane of guinea pig kidney proximal tubule suggests that melatonin plays a role in regulating epithelial functions. To investigate the cellular basis of melatonin action on epithelia, we sought to establish an appropriate in vitro culture model. Epithelial cell lines originating from kidneys of dog (MDCK), pig (LLC-PK1), opossum (OK), and human embryo (HEK- 293) were each tested for the presence of MR using 2-[125I]iodomelatonin (125I-MEL) as a radioligand. The HEK-293 cell line exhibited the highest specific 125I-MEL binding. By intermediate filament characterization, the HEK-293 cells were determined to be of epithelial origin. Binding of 125I- MEL in HEK-293 cells demonstrated saturability, reversibility, and high specificity with an equilibrium dissociation constant (K(d)) value of 23.8 ± 0.5 pM and a maximum number of binding sites (B(max)) value of 1.17 ± 0.11 fmol/mg protein (n = 5), which are comparable with the reported K(d) and B(max) values in human kidney cortex. Coincubation with GTPyS (10 μM) and pertussis toxin (100 ng/ml) provoked a marked decrease in binding affinity (K(d) was increased by a factor of 1.5-2.0), with no significant difference in B(max). Melatonin (1 μM) decreased the forskolin (10 μM) stimulated cAMP level by 50%. HEK-293 cells do not express dopamine D1A receptor. Following transient transfection of HEK-293 cells with human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamine stimulated an increase in the level of cAMP. Similarly, transient transfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and PTH type 1 receptors, each resulted in an hormone inducible increase in cAMP levels. Surprisingly, only the stimulatory effect of dopamine could be inhibited by exposure to melatonin. The inhibitory effect of melatonin on dopamine D1-induced increase in cAMP was completely inhibited by pertussis toxin (100 ng/ml, 18 h). Immunoblot and immunocytochemical studies were carried out using two polyclonal antibodies raised against the extra and cytoplasmic domains of Mel receptor. Immunoblot studies using antibody against the cytoplasmic domain of Mel(1a) receptor confirmed the presence of a peptide blockable 37 kDa band in HEK-293 cells. Indirect immunofluorescent studies with both antibodies revealed staining predominantly at the cell surface, but staining with the antibody directed against the cytoplasmic domain required prior cell permeabilization. By RT- PCR, HEK-293 cells express both Mel(1a) and Mel(1b) messenger RNAs, but the messenger RNA level for Mel(1b) is several orders of magnitude lower than for Mel(1a). We conclude that HEK-293 cells express MR predominantly of the Mel(1a) subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is through modulation of cellular dopaminergic responses.published_or_final_versio