Mutations of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to cystic fibrosis, an autosomal recessive genetic disorder affecting a number of organs including the lung airways, pancreas and sweat glands. In order to investigate the post-translational ligation of CFTR with reconstructed functional chloride ion channel and the split Ssp DnaB intein-mediated protein trans-splicing was explored to co-deliver CFTR gene into eukaryotic cells with two vectors. The human CFTR cDNA was split after Glu838 codon before the second transmembrane dome (TMD2) into two halves of N- and C-parts and fused with the coding sequences of split Ssp DnaB intein. Pair of eukaryotic expression vectors pEGFP-NInt and pEYFP-IntC were constructed by inserting them into the vectors pEGFP-N1 and pEYFP-N1 respectively. The transient expression was carried out for observing the ligation of CFTR by Western blotting and recording the chloride current by patch clamps when cotransfection of the pair of vectors into baby hamster kidney (BHK) cells. The results showed that an obvious protein band proven to be ligated intact CFTR can be seen and a higher chloride current and activity of chloride channel were recorded after cotransfection. These data demonstrated that split Ssp DnaB intein could be used as a strategy in delivering CFTR gene by two vectors providing evidence for application of dual adeno-associated virus (AAV) vectors to overcome the limitation of packaging size in cystic fibrosis gene therapy.
Animals ; Cell Line ; Chloride Channels ; physiology ; Cricetinae ; Cystic Fibrosis Transmembrane Conductance Regulator ; biosynthesis ; genetics ; Dependovirus ; genetics ; Genetic Vectors ; Humans ; Membrane Potentials ; genetics ; Protein Processing, Post-Translational ; Protein Splicing

deltaFosB, a naturally occurring truncated isform of fosB gene, existed in many tissues stably and played an important role in formation and differentiation of adipocyte and osteoblast. deltaFosB may be related to the metabolism of calcium in bone and mammary gland and regulate the signal pathway of calcium transfer from bone to mammary gland. We first sub-cloned deltafosB gene of goat into the vector pET32a to construct prokaryotic expression vector pET32a-deltafosB. Then we induced for deltafosB gene expression efficiently by IPTG. Finally we immunized the adult rabbits with purified recombinant deltaFosB to prepare rabbit anti-goat deltaFosB polyclonal antibody. iELISA analysis showed the antibody with the titer of 1:51 200, and Western blotting result showed that the antibody could specifically detect the deltaFosB protein expressed in prokaryotic cell and HEK-293 cell, respectively. Further Western blotting assay showed that deltaFosB expressed in various tissues of goat in vivo.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Goats ; Proto-Oncogene Proteins c-fos ; biosynthesis ; genetics ; immunology ; Rabbits ; Recombinant Proteins ; biosynthesis ; genetics ; immunology

Homologous recombination is an important technique that is used to modify mammalian genome. Here, we constructed an efficient common gene targeting vector based on the plasmid pBS246. The vector consisted two positive selection markers, neomycin resistance gene (neo) and enhanced green fluorescent protein gene (EGFP) flanked by locus of X-over P1 (LoxP) sites. Two synthesized multiple cloning sequences MCS-1 and MCS-2 that contain several "8 bp cutter" enzyme sites were placed in outside of LoxP sites. Additionally, a negative selection marker HSV-tk (herpes simplex virus thymidine kinase) gene was located adjacent to MCS-1 site. The constructed vector was named pGT-V1, and its functions were characterized in C2C12 cells. The vector had the following unique features: 1) EGFP was used to monitor instantly the transfection rate that was essential for increasing the efficiency of gene knockout (KO); 2) The EGFP marker located between two LoxP sites was able to be removed from KO positive cells to avoid the potential damage of selection markers to the recipient cells. The process could be monitored visually and the positive cells without selecting markers (the loss of green fluorescent cells) could be sorted out by either flow cytometry or immunomagnetic beads; 3) "8 bp cutter" restriction sites were embedded in MCS sequences, which then enhanced the versatility of this vector. In summary, the constructed plasmid optimized the vector of gene targeting and provided a new technique means for the transgenic animal research.
Animals ; Base Sequence ; Cloning, Molecular ; Drug Resistance ; genetics ; Gene Knockout Techniques ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Homologous Recombination ; Mice ; Molecular Sequence Data ; Neomycin ; pharmacology

Generation of bio-engineered teeth by using stem cells will be a major approach for bioengineered implantation. Previous studies have demonstrated that dissociated tooth germ cells are capable of generating a tooth after reaggregation in vitro. However, the cellular and molecular mechanisms underlying this tooth regeneration are not clear. In this study, we dispersed E13.5 molar germ into single cells, immediately reaggregated them into cell pellet, then grafted the reaggregates under mouse kidney capsule for various times of culture. We investigated the morphogenesis and the expression of several developmental genes in dental epithelial cells in reaggregates of tooth germ cells. We found that dissociated tooth germ cells, after reaggregation, recapitulated normal tooth developmental process. In addition, dissociated dental epithelial cells retained the expression of Fgf8, Noggin, and Shh during reaggregation and tooth regeneration processes. Our results demonstrated that, despite of under dissociated status, dental epithelial cells maintained their odontogenic fate after re-aggregation with dental mesenchymal cells. These results provided important information for future in vitro generation of bio-engineered teeth from stem cells.
Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Embryo, Mammalian ; Epithelial Cells ; cytology ; metabolism ; Female ; Gene Expression Regulation, Developmental ; genetics ; Male ; Mice ; Odontogenesis ; genetics ; Tooth Germ ; cytology ; physiology

To investigate the effect of the localization of oligonucleotides decoy (ODNs decoy) on the activation of nuclear factor-kappaB (NF-kappaB) in TNF-alpha induced HeLa cells. The mercapto group-modified nuclear localization signal (NLS) peptide was covalently conjugated to amino group-modified NF-kappaB ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy was transfected into HeLa cells by TransME transfection reagent. The intracellular distribution of fluorescent labeled NLS-ODNs decoy was detected with a microscope. The cell viability was detected by MTT assay, and then the activity of NF-kappaB in cell nuclear extract was assayed by electrophoretic mobility shift assay (EMSA). The results showed that NLS peptide was successfully conjugated to ODNs decoy by Sulfo-SMCC cross-linker. The NLS-ODNs decoy effectively entered into nucleus with high rate of 17.9%. It was observed that the cell viability of HeLa cell was not significantly affected by the transfection of NLS-ODNs decoy, while NLS-ODNs decoy significantly inhibited the activation of NF-kappaB in TNF-alpha induced HeLa cells nuclear extracts. This experiment can provide a new covalent conjugation of NLS peptide to ODNs can effectively drive decoy into nucleus, and thus improve its inhibitory effects on the activation a transcription factor.
Base Sequence ; Cell Nucleus ; metabolism ; HeLa Cells ; Humans ; Molecular Sequence Data ; NF-kappa B ; genetics ; metabolism ; Nuclear Localization Signals ; genetics ; Oligonucleotides ; genetics ; metabolism ; Transfection

As a virus-like particle, hepatitis B surface antigen (HBsAg) was the primary component of hepatitis B vaccine. HBsAg was maintained by the non-covalent interaction of proteins and lipids. The intact structure of HBsAg particle was vital to its function. However, there was no report about the effects of solvent environment on HBsAg structure. In this paper, we studied the effects of temperature, pH, ionic type and salt concentration on HBsAg structure. The results showed that HBsAg was stable at normal temperature, but began to denature above 60 degrees C. The aggregation of HBsAg at pH 3.0 and 4.0 was nearly irreversible, but partly reversible at pH 5.0. The influence of ionic type on HBsAg was generally in accordance with Hofmeister sequence, except that SO4(2-) caused more aggregation than F-. HBsAg aggregates started to be visible in 0.4 mol/L (NH4)2SO4, and the extent of aggregation increased with the salt concentration. Therefore, caution must be taken when using (NH4)2SO4 in the hydrophobic chromatography purification of HBsAg.
Ammonium Sulfate ; chemistry ; Hepatitis B Surface Antigens ; chemistry ; Hepatitis B Vaccines ; chemistry ; Hepatitis B virus ; chemistry ; Hydrogen-Ion Concentration ; Protein Denaturation ; Solvents ; Temperature

The Forkhead box O1 (FoxO1) transcription factor governs muscle growth, metabolism and cell differentiation. However, its role in myoblast differentiation is unclear. To study the biological function of FoxO1 during differentiation in porcine primary myoblast, we constructed stably FoxO1 over-expressed porcine myoblast mediated by liposome and adopted morphological observation, quantitative real-time RT-PCR and Western blotting methods to analyze FoxO1 and early and late myogenic regulation factors MyoD and myogenin expression. During differentiation the mRNA level of FoxO1 was significantly increased. However, the total protein did not change but the phosphorylation of FoxO1 was upregulated. Furthermore, overexpression of FoxO1 in porcine myoblast decreased MyoD and myogenin mRNA, whereas MyoD protein changed little and myogenin was significantly suppressed (P < 0.05). These results indicated that FoxO1 delays and negatively regulates the porcine myoblast differentiation. Moreover, FoxO1 may play a critical role in muscle fiber-type specification through the inhibition of myogenic regulation factors.
Animals ; Animals, Newborn ; Cell Differentiation ; genetics ; Cells, Cultured ; Forkhead Transcription Factors ; biosynthesis ; genetics ; Muscle, Skeletal ; cytology ; metabolism ; Myoblasts ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Swine

Currently, animal somatic cell reprogramming into the induced pluripotent stem cell (iPS) is one of the hottest research target in the field of cell biology. We focused on the analysis of telomerase reverse transcriptase (TERT) gene expression during goat somatic fibroblasts reprogramming, and investigated the relationship between the expression of TERT and the pluripotency of reprogrammed cells. RNA samples of fetal tissues isolated from Guanzhong milk goat fetus, and the induced goat reprogramming cell clones were used to determine the relative expression levels of TERT by the real-time RT-PCR method. Goat embryonic fibroblasts (GEF) collected from the Guanzhong milk goat with normal karyotype were induced by 4 transcription factors to become reprogramming cells. The expression of TERT in reprogramming cells was detected by Real-time RT-PCR. The results showed that the expression of TERT in testis tissue was higher than that in epithelial tissues (P < 0.01). The expression level of TERT was higher in AP staining positive cells than that in AP staining negative cells (P < 0.01). This result indicated that TERT activity played an important role in cell reprogramming.
Animals ; Cellular Reprogramming ; Fibroblasts ; cytology ; Gene Expression Regulation ; Goats ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; RNA-Directed DNA Polymerase ; genetics ; Telomerase ; metabolism

In order to study PBD-2 and PoIFNgamma, the chimeric gene PBD-2-PoIFNgamma was synthesized by overlap extension PCR, and amplified PoIFNgamma on the basis of this sequence, then cloned into yeast expression vector pPICZalphaA separately to get the recombinant plasmid pPICZalphaA-PBD-2-PoINFgamma and pPICZalphaA-PoINFgamma. The recombinant plasmid was digested by Sac I and introduced into Pichia pastoris X33 cells by electroporation. Positive clones were screened and cultivated in BMMY medium containing 0.5% methanol for 72 h. SDS-PAGE and Western blotting analysis showed that the screened recombinant could secrete PBD-2-PoINFgamma and PoINFgamma separately. The activity of fusion protein was not detected by cytopathic effect inhibition assay and agar diffusion assay, but detected obvious antiviral activity of PoINFgamma. The helix and random coil contents was showed vary greatly between PoIFNgamma and PBD-2-PoLNFgamma by circular dichroism analysis. It was speculated that the fusion protein was not correctly folded and may affect the activity of PBD-2-PoINFgamma.
Animals ; Genetic Vectors ; genetics ; Interferon-gamma ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Swine ; beta-Defensins ; biosynthesis ; genetics

To obtain bovine adipose-derived stem cells (ADSCs), bovine ADSCs were digested in collagenase type I solution. The growth curve of ADSCs was checked by cell counting. Chromosome analysis was checked. The molecular markers of ADSCs were detected with immunofluorescence staining. The morphology of ADSCs was identical to fibroblast like and the cells showed active proliferative ability. Vimentin, CD49d and CD13 antigens were detected, but CD34 antigen was negative. Alkaline phosphatase activity was greater in ADSCs during calcification, and Alizarin Red staining was positive. Lipid droplets were apparent around cells during adipogenesis, and Oil Red-O staining was positive. The results demonstrated that ADSCs could be used as seed cells for tissue engineering due to the simple isolation, differentiation and stable and active growth.
Adipose Tissue ; cytology ; Animals ; Cattle ; Cell Differentiation ; physiology ; Cell Separation ; Cells, Cultured ; Mesenchymal Stromal Cells ; cytology ; Tissue Engineering ; methods