Pyrroloquinoline quinone (PQQ), an important redox enzyme cofactor, has many physiological and biochemical functions, and is widely used in food, medicine, health and agriculture industry. In this study, PQQ production by recombinant Gluconobacter oxydans was investigated. First, to reduce the by-product of acetic acid, the recombinant strain G. oxydans T1 was constructed, in which the pyruvate decarboxylase (GOX1081) was knocked out. Then the pqqABCDE gene cluster and tldD gene were fused under the control of endogenous constitutive promoter P0169, to generate the recombinant strain G. oxydans T2. Finally, the medium composition and fermentation conditions were optimized. The biomass of G. oxydans T1 and G. oxydans T2 were increased by 43.02% and 38.76% respectively, and the PQQ production was 4.82 and 20.5 times higher than that of the wild strain, respectively. Furthermore, the carbon sources and culture conditions of G. oxydans T2 were optimized, resulting in a final PQQ yield of (51.32±0.899 7 mg/L), 345.6 times higher than that of the wild strain. In all, the biomass of G. oxydans and the yield of PQQ can be effectively increased by genetic engineering.
Fermentation ; Gene Knockout Techniques ; Gluconobacter oxydans ; genetics ; metabolism ; Industrial Microbiology ; methods ; Multigene Family ; genetics ; Organisms, Genetically Modified ; PQQ Cofactor ; biosynthesis ; genetics ; Promoter Regions, Genetic ; genetics

Trehalose, a compatible solute, is widely used in food, cosmetics, pharmaceutical products and organ transplantation. Nowadays, trehalose is mostly produced by enzymatic synthesis with many secondary products and lowpurity. In this study, high amount of trehalose was produced by recombinant E. ccli fermentation. First, a bifunctional trehalose gene TPSP was amplified from genome of C. hutchinscoii. Second, an expression vector pTac-HisA containing TPSP was constructed and transformed into the host E. coli. Expression of this bifunctional enzyme-TPSP converted glucose to trehalose. The result suggested that TPSP from C. hutchinsonji has been successfully expressed in E. ccoi. High amount of extracellular trehalose generated from glucose by whole-cell catalysis and After optimization, the production of trehalose in shake flasks was improved to 1.2 g/L and the relative conversion rate reached 21%. The production in bioreactor reached 13.3 g/L and the relative conversion rate reached 48.6%. It is the first time to realize the functional expression of the bifunctional enzyme-TPSP of C. hutchinsonii in E. coli and achieved the conversion form glucose to trehalose. This study laid a foundation for industrial large-scale production of trehalose.
Bioreactors ; Catalysis ; Escherichia coli ; genetics ; Glucose ; Glucosyltransferases ; Industrial Microbiology ; Organisms, Genetically Modified ; Trehalose ; biosynthesis

This study was aimed to investigate the homing capacity of CXCR4 overexpressed mesenchymal stem cells (MSC) and their effect on hematopoietic recovery. The 293FT packaging cell line was transfected with the recombinant lentiviral vector LV-CXCR4-IRES-EGFP and LV-IRES-EGFP to produce lentivirus. Mouse MSC were then infected with viral supernatant. Male BALB/c mice were sublethally irradiated and then were injected intravenously with 5×10(5) MSC. General status and survival rate of mice were observed every day. On day 3, 7, 14, 21 and 28, peripheral blood samples were collected to calculate the number of white blood cells (WBC) and red blood cells (RBC), the ratio of reticulocyte to platelet, the number of platelet was detected by flow cytometry. The recovery of bone marrow and spleen was pathologically monitored. The proportion of MSC implantation was analysed by PCR. The results showed that the peripheral blood cells displayed the tendency of firstly increasing and then decreasing to their normal level. Generally, recovery of WBC level was earlier in mice infused with MSC (P < 0.05) . The histopathological examination of spleen and bone marrow showed a faster hematopoietic recovery in CXCR4-MSC group than the other two groups. And the donor MSC could be detected in the recipients on day 7, 14, 21 and 28. It is concluded that infusion of CXCR4-MSC enhances the implantation of hematopoietic stem cells and promotes hematopoietic recovery of the sublethally irradiated mice.
Animals ; Bone Marrow Cells ; cytology ; Genetic Vectors ; Hematopoiesis ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Organisms, Genetically Modified ; Radiation Injuries, Experimental ; therapy ; Receptors, CXCR4 ; genetics ; Transfection ; Whole-Body Irradiation

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.
Amebicides/*pharmacology ; Animals ; Flow Cytometry ; Gentamicins/*pharmacology ; Green Fluorescent Proteins/*chemistry ; Host-Parasite Interactions ; Leishmania mexicana/drug effects/genetics/*growth & development ; Leishmaniasis, Cutaneous/*parasitology ; Luminescent Agents/*chemistry ; Macrophages, Peritoneal/parasitology ; Mice ; Mice, Inbred BALB C ; Organisms, Genetically Modified ; Spectrometry, Fluorescence

Th-2-biased immune responses are known to play a key role in the pathogenesis of atopic dermatitis. In particular, the macrophage-derived chemokine CCL22 is directly implicated in Th-2-associated skin inflammatory reactions, and its levels are significantly elevated in serum and are correlated with disease severity in atopic dermatitis. In this study, we tested the development of genetic therapeutic options to treat atopic dermatitis using bacteria expressing miRNA. We constructed a recombinant strain of Salmonella typhimurium expressing CCL22 miRNA (ST-miRCCL22) for the in vivo knockdown of CCL22. The CCL22 gene was downregulated with CCL22 miRNA in activated lymphocytes. In mice with a cutaneous disease similar to atopic dermatitis, interleukin-4 was inhibited and interferon-gamma was induced after treatments with ST-miRCCL22. Furthermore, CCL22 levels were suppressed in the atopic mice treated with ST-miRCCL22. These results suggest that ST-miRCCL22 may be an effective genetic agent for treating atopic dermatitis.
Animals ; Cell Line ; *Chemokine CCL22/genetics ; Cytokines/blood ; Dermatitis, Atopic/pathology ; Disease Models, Animal ; Female ; Gene Expression Regulation/drug effects ; Gene Silencing ; Immunoglobulin E/blood ; Mice ; *MicroRNAs/genetics/pharmacology ; *Organisms, Genetically Modified/genetics ; *Salmonella typhimurium/genetics/metabolism ; Skin/*drug effects/pathology

This study was aimed to investigate the protective effect of Wit3a gene modification on mouse bone marrow mesenchymal stem cells against the injury induced by Ara-C. The gene-modified MSC steadily expressing Wnt3a were established by adenovirus system. The acute direct damage effects of different concentrations of Ara-C on the unmodified MSC and the gene-modified MSC were assessed by using an in vitro culture system, and the corresponding controls were set. The proliferation and apoptosis of MSC exposed to Ara-C were detected by cell count kit-8 (CCK-8) and flow cytometry. The expression of BCL-2 protein related with cell apoptosis was assayed by Western blot. The results indicated that as compared with unmodified MSC, Ara-C exhibited a less inhibitory effect on the proliferation of gene-modified MSC. There was obvious difference between unmodified MSC and gene-modified MSC (p < 0.05). The proliferation of gene-modified MSC began to recover at 72 hours after removal of Ara-C. However, unmodified MSC showed sustained suppression of proliferation after withdrawal of Ara-C. In apoptosis, the apoptosis rate of gene-modified MSC induced by Ara-C was significantly lower than those of unmodified MSC (p < 0.05). In addition, the expression levels of BCL-2 protein in gene-modified MSC were up-regulated compared with unmodified MSC (p < 0.05). It is concluded that Wnt3a gene modification can significantly mitigate the damage of mouse bone marrow MSC induced by Ara-C.
Animals ; Bone Marrow Cells ; drug effects ; metabolism ; Cytarabine ; adverse effects ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; Mice ; Organisms, Genetically Modified ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Wnt3A Protein ; genetics

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Proliferation ; Interleukin-10 ; genetics ; metabolism ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Organisms, Genetically Modified ; Rats ; Rats, Sprague-Dawley ; Transfection

The study was aimed to investigate the effect of deriving hematopoietic cells from human embryonic stem cells (hESCs) by the erythropoietin gene-modified conditioned medium of human mesenchymal cells. The mesenchymal stem cells (MSCs) steadily expressing EPO were established by lentiviral system. The expression of exogenous EPO was detected by RT-PCR and Western blot. After suspension culture, hESCs developed into embryonic bodies (EBs). Then the EB cells were cultured in conditional medium. The hESCs-derived hematopoietic cells were analyzed by immunofluorescence, CFU assay and RT-PCR. The results indicated that the exogenous EPO successfully expressed in the EPO transfected MSCs (EPO/MSCs). The supernatant from EPO/MSCs increased CD34(+) cell population and the expression of globin, and enhanced colony forming unit incidence. These effects were obviously higher than that of control. It is concluded that the EPO gene-modified conditioned medium of human mesenchymal cells can induce the hESCs to differentiate into hematopoietic cells.
Cell Culture Techniques ; Cell Differentiation ; drug effects ; Culture Media, Conditioned ; pharmacology ; Embryonic Stem Cells ; cytology ; drug effects ; Erythropoietin ; genetics ; pharmacology ; Hematopoietic System ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Organisms, Genetically Modified

To construct the recombinant vector Pact-EGFP, the Act-1 core promoter region was amplified from the pUCm-T/Act-1 and subcloned into pEGFP-4.1 vector (derived from pEGFP-N1 with the removal of human cytomegalovirus immediate early promoter), by restriction enzymes Bgl II and Hind III. Transfection of Pact-EGFP vector into Vero cell by liposome indicated that Act-1 core promoter regulated the expression of EGFP gene in lower level in Vero cells. After Pact-EGFP microinjection into the gonad of Caenorhabditis elegans with pRF4 as a gene marker, green fluorescence was detected in the cortex, vice cortex and the pharyngeal of C. elegans. According to the locations, two different transgene lines were separated. The expression level of EGFP expressed in C. elegans was more than that in Vero cell. Some unique motifs might exist in Act-1 core promoter region of C. elegans, which was closely related to the expression level of EGFP. These results lay the foundation for the further research on gene function of parasitic nematodes using C. elegans.
Animals ; Caenorhabditis elegans ; genetics ; metabolism ; Cercopithecus aethiops ; Connexin 43 ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Organisms, Genetically Modified ; Peptide Fragments ; genetics ; Promoter Regions, Genetic ; genetics ; Transcription, Genetic ; Vero Cells

To optimize the electroporation system in Dunaliella salina (D. salina), we studied the effects of growth phase of cells, electric parameters, electroporation buffer and concentration of plasmid on transformation efficiency. The results showed that a transformation efficiency of 1.81 per thousand was achieved in D. salina cells at mid-log growth phase electroporated with plasmid (DCA-bar) 10 microg/mL, voltage 0.8 kV and capacitance 25 microF. However, when glycerol was added to electroporation buffer at a final concentration of 0.4 mol/L, the transformation efficiency was increased up to 2.03 per thousand. Additionally, transformation was done with plasmids DCA-bar, NR-bar, pUomega-bar respectively, under above optimum conditions, and similar transformation efficiencies were obtained. The findings indicate that an efficient and stable system of electroporation in D. salina has been developed, providing a powerful tool for the transgenic research of D. salina.
Chlorophyta ; cytology ; genetics ; Culture Media ; Electroporation ; Organisms, Genetically Modified ; genetics ; Transformation, Genetic ; genetics