ABSTRACT
Objective: To investigate the overexpression of paired box gene 6(Pax6) gene in mouse embryonic stem cells and obtain cell line, which is the basis for further differentiation of Pax6/mouse embryonic stem cells (mESCs). Methods: mESCs were cultured in vitro, and the recombinant vector pEFla-Pax6-IRES-AcGFP and the empty vector pEFlot-IRES-AcGFP were transfected into mESCs by liposome method respectively. The cells were screened by G418 gradient and fluorescent protein. The expression of Pax6 was detected by RT-PCR, immunofluorescence and Western blotting and the proportion of Pax6/mESCs positive cells was detected by flow cytometry. The obtained cell line was detected by cell immunofluorescence for stem cell markers stage specific embryonic antigen 1 (SSEA1) and octamer binding transcription factor 4(0CT4), and the pluripotency was detected by alkaline phosphatase staining. Pax6/mESCs cells were subcutaneously transplanted, and the grafts were observed by HE staining to observe their differentiation ability. Results: Pax6 was successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs was obtained, and the flow rate showed positive rate about 90%. Immunofluorescence showed that stem cell markers SSEA1 and 0CT4 were positively expressed and alkaline phosphatase stained cells were stained brownish black, and transplantation in vivo could differentiate into three germ layers. Conclusion: Pax6 is successfully expressed in mESCs. After screening by G418, the cell line Pax6/mESCs is obtained and shows the good stem cell characteristics.
ABSTRACT
Pectate lyase is widely applied in ramie degumming and fabric bioscouring in the textile industry. Compared to conventional processes that involve high alkaline and high temperature treatment, enzyme based treatments have significant advantages in fibers protectiveness, improved efficiency of refining, reduced energy consumption and pollution. Hence, it would be highly desirable to construct high-yield alkaline pectate lyase engineered strains and reduce the pectate lyase production cost. In the previous study, pectate lyase gene pel from Bacillus subtilis168 was expressed in Pichia pastoris GS115 after codon usage optimization based on the vector pHBM905A. To improve the expression level, the vector pHBM905BDM with optimized promoter and signal peptide was used to express the optimized gene pels in GS115. The transformant had increased activity from 68 U/mL to 100 U/mL with the improvement in the transcription level by 27% measured by qPCR. The transformants were further screened on pectin plates, where higher halo forming strains were picked for shake-flask fermentation and strain GS115-pHBM905BDM-pels4 showed the highest activity of 536 U/mL. Then plasmid pPIC9K-pels was constructed and electroporated into the GS115-pHBM905BDM-pels4 cells. Subsequently, high-copy transformant was screened by using the medium containing antibiotics G418, strain GS115-pHBM905BDMpPIC9K- pels1 was identified with increased activity of 770 U/mL and the copy number of pels was 7 confirmed by qPCR. Finally, the activity of pectate lyase produced by GS115-pHBM905BDM-pPIC9K-pels1reached to 2 271 U/mL in a 5-L fermentor. The activity of pectate lyase in our study reached the highest level of expression in P. pastoris, showing good application potential in the textile industry.
ABSTRACT
Aim To construct the stable hFcεRIα/RBL-2H3 cell line expressing human FcεRIα( hFcεRIα) . Methods The human FcεRIα gene was obtained by RT-PCR and cloned into the eukaryotic expression vector pCI-neo. Then, the pCI-neo-hFcεRIα vector was transfected into RBL-2H3 cells by lipo-somes, and the transfected cells were screened through G418 fil-tration subsequently. Finally, RT-PCR, Western blot and immu-nofluorescence assay were used to determine the result of trans-fection. Results According to the optimized transfection param-eters, the transfection efficiency reached 75. 38%. The results of Western blot, immunofluorescence and RT-PCR showed that hFcεRIα could be expressed in RBL-2H3 cells successfully. Conclusion HFcεRIα/RBL-2H3 cells were successfully con-structed,which will be the experimental basis for further study on the mechanism of IgE/FcεRI and drugs for allergy diseases.
ABSTRACT
Glucose was transported by the large number of hexose transporters in yeast cells. There were 18 hexose transporter genes had been identified in Saccharomyces cerevisiae. However,as an excellent expression system,there was no information of these genes had been reported in Pichia pastoris. Based on high homologous recombination efficiency in yeast,we chose G418 resistance for screening,200 bp were cloned from the up and down sequences of HXT1 ORF respectively,then ligated to the 5′ and 3′ end of G418 resis-tance gene for recombination. After electroporation of GS115 spheroplast and screened through different G418 concentration plates,finally we obtained one HXT1 gene deletion mutant named GS115?HXT1. The growth rate and glucose consumption of this mutant were both lower than the wide type.
ABSTRACT
PURPOSE: Some antibiotics were known to exert neuroprotective effects in the animal model of hypoxic-ischemic (H-I) brain injury, but the mechanism is still unclear. A recent study reported that geneticin (G418), an aminoglycoside antibiotic, increased survival of human breast cancer cells by suppressing apoptosis. We investigated the neuroprotective effects of systemically administrated geneticin via anti-apoptosis following the H-I brain injury METHODS: Seven-day-old Sprague-Dawley rat pups were subjected to unilateral (left) common carotid artery occlusion followed by 2.5 hours of hypoxic exposure and the cortical cell culture of rat brain was done under a hypoxic incubator. Apoptosis was measured in the injured hemispheres 7 days after H-I insult and in the injured cells from hypoxic chamber using morphologic analysis by Terminal dUTP Nick-end Labeling(TUNEL) assay and immunohistochemistry for caspase-3, and cytologic analysis by western blot and real time PCR for bax, bcl-2, and caspase-3. RESULTS: The gross appearance and hematoxylin and eosin stain revealed increased brain volume in the geneticin-treated animal model of perinatal H-I brain injury. The TUNEL assay revealed decreased apoptotic cells after administration of geneticin in the cell culture model of anoxia. Immunohistochemistry showed decreased caspase-3 expression in geneticin-treated cortical cell culture. Western blot and real-time PCR showed decreased caspase-3 expression and decreased ratio of Bax/Bcl-2 expression in geneticin-treated animal model. CONCLUSION: Geneticin appears to exert a neuroprotective effect against perinatal H-I brain injury at least via anti-apoptosis. However, more experiments are needed in order to demonstrate the usefulness of geneticin as a preventive and rescue treatment for H-I brain injuries of neonatal brain.
Subject(s)
Animals , Humans , Rats , Hypoxia , Anti-Bacterial Agents , Apoptosis , Blotting, Western , Brain , Brain Injuries , Breast Neoplasms , Carotid Artery, Common , Caspase 3 , Cell Culture Techniques , Eosine Yellowish-(YS) , Gentamicins , Hematoxylin , Immunohistochemistry , In Situ Nick-End Labeling , Incubators , Models, Animal , Neuroprotective Agents , Real-Time Polymerase Chain ReactionABSTRACT
PURPOSE: Recently, Geneticin (G418) were known to exert neuroprotective effects in the hypoxic-ischemic (H-I) brain injury, but the mechanism is still unclear. The roles of fibroblast growth factor (FGF) and FGF receptor (FGFR) ware not well known in the H-I brain injury. We investigated the neuroprotective effects of systemically administrated Geneticin through the regulation of FGFR following the H-I brain injury METHODS: The cortical neuron cell culture of Spague-Dawley (SD) rat embryo brain (E18) was done in a hypoxic incubator. The cultured cells were divided three groups: a normoxia group, a hypoxia group, and an Geneticin-treated group. After verifying the desired amount of cellular injury in the hypoxia group, the Geneticin-treated group (after an H-I insult) was further divided into two groups. This produced four final groups: normoxia, hypoxia, and Geneticin-treated groups before H-I insult and a Geneticin-treated group after HI insult. The expression of FGFR-2 and FGFR-3 mRNA was measured using Northern blotting. RESULTS: The expression of FGFR-2 and FGFR-3 mRNA was notably increased in the hypoxic group compared to the normoxic group. In both Geneticin-treated groups before and after a hypoxic insult, the expression of FGFR-2 and FGFR-3 mRNA was decreased. CONCLUSION: It suggests that FGFR has an important role in hypoxic brain injury. Geneticin appears to exert a protective effect through down regulation of the expression of FGFR mRNA. However, more experiments are needed in order to demonstrate the usefulness of Geneticin as a preventative and rescue treatment for H-I brain injuries of neonatal brain.