RESUMO
Glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, and NAGA) play an important regulatory role in the defense against Escherichia coli F18 in piglets. In this study, we identified the transcription initiation site and promoter of this gene cluster by mined previous RNA-seq results using bioinformatics tools. The FUT1 transcription initiation region included five alternative splicing sites and two promoter regions, whereas each of the six other genes had one promoter. Dual luciferase reporter results revealed significantly higher transcriptional activity by FUT1 promoter 2, indicating that it played a more important role in transcription. The promoters of glycosphingolipid biosynthesis genes identified contained a CpG island within the first 500 bp, except for the B3GALNT1 promoter which included fewer CpG sites. These results provide a deeper insight into methylation and the regulatory mechanisms of glycosphingolipid biosynthesis-globo series pathway genes in piglets.
Assuntos
Fucosiltransferases/genética , Glicoesfingolipídeos/biossíntese , Regiões Promotoras Genéticas , Suínos/genética , Animais , Ilhas de CpG , Metilação de DNA , Fucosiltransferases/metabolismo , Ativação Transcricional , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Preliminary studies have suggested that a characteristic element of the matrix attachment region (MAR) in human interferon-ß mediates the adhesion of vectors to Chinese hamster ovary (CHO) cells. In this study, we investigated if vector adhesion increased nerve growth factor (NGF) expression in CHO cells. The MAR characteristic element sequence of human interferon-ß was inserted into the multiple-cloning site of the pEGFP-C1 vector. The target NGF gene was inserted upstream of the MAR characteristic element sequence to construct the MAR/NGF expression vector. The recombinant plasmid was transfected into CHO cells and stable monoclonal cells were selected using G418. NGF mRNA and protein expression was detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Plasmid reduction experiments were used to determine the state of transfected plasmid in mammalian cells. The insertion of MAR into the vector increased NGF expression levels in CHO cells (1.93- fold) compared to the control. The recombinant plasmid expressing the MAR sequence was digested into a linear space vector. The inserted MAR and NGF sequences were consistent with those inserted into the plasmid before recombination. Therefore, we concluded that the MAR characteristic element mediates vector adhesion to CHO cells and enhances the stability and efficiency of the target gene expression.
Assuntos
Regulação da Expressão Gênica , Vetores Genéticos/genética , Regiões de Interação com a Matriz , Fator de Crescimento Neural/genética , Animais , Células CHO , Cricetulus , Ordem dos Genes , Plasmídeos/genéticaRESUMO
We investigated the effects of different directions of insertion of matrix attachment region (MAR) sequences on transgenic expression in stably transformed Chinese hamster ovary (CHO) cells. The MAR sequences were inserted in forward or reverse directions into the expression vectors, and transfected into CHO cells. The expression of the chloramphenicol acetyltransferase (CAT) reporter gene and the relative copy numbers of the CAT gene were analyzed. The CAT gene expression levels in the vector with the MAR sequence inserted in the forward or reverse directions increased compared with expression without the MAR sequence. The relative copy numbers of the CAT gene with MAR sequenced vectors inserted in the reverse and forward directions were lower, than in the control group. The direction of insertion of MAR sequences had no significant effect on expression levels. The expression levels were not proportional to the copy numbers of the gene.
Assuntos
Cloranfenicol O-Acetiltransferase/genética , DNA Intergênico/genética , Vetores Genéticos/química , Regiões de Interação com a Matriz , Plasmídeos/química , Animais , Células CHO , Linhagem Celular Transformada , Cloranfenicol O-Acetiltransferase/metabolismo , Cricetulus , DNA Intergênico/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/metabolismo , Plasmídeos/metabolismo , TransgenesRESUMO
The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.
Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Proteínas Sanguíneas/biossíntese , Resistência à Doença/genética , Infecções por Escherichia coli/genética , Escherichia coli/genética , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Sanguíneas/genética , Suscetibilidade a Doenças/microbiologia , Suscetibilidade a Doenças/veterinária , Endotoxinas/genética , Endotoxinas/metabolismo , Escherichia coli/patogenicidade , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Infecções por Escherichia coli/veterinária , Vetores Genéticos , Genótipo , Suínos , DesmameRESUMO
The transporter associated with antigen processing (TAP) transports peptides from the cytosol into the endoplasmic reticulum for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. This study showed the dynamic changes in the TAP1 expression level in newborn to weaning piglets. Tissue expression profiles revealed that the TAP1 gene was expressed at low levels in all tissues, and the expression levels were relatively higher in the lung, spleen, lymph, and thymus; further, no significant difference was observed in the expression in each tissue among the 3 unweaned stages (8, 18, and 30 days). Nevertheless, the postweaning (35 days) expression levels in tissues, including the spleen, lung, lymph, duodenum, and jejunum were significantly higher than those in the unweaned stages. Furthermore, gene ontology and pathway analysis showed that TAP1 took part in 38 biological functions and 5 pathway processes, including ABC transporters and antigen processing and presentation. These analyses showed that the TAP1 gene, which was related to MHC I immune regulation, had a stable and low expression level in unweaned stages; however, its expression increased in the postweaning stages. The high expression level of TAP1 indicated that the gene might play an important role in Escherichia coli F18 resistance.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Suínos/genética , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Escherichia coli/patogenicidade , Regulação da Expressão Gênica no Desenvolvimento , Antígenos de Histocompatibilidade Classe I/biossíntese , Suínos/crescimento & desenvolvimento , Suínos/microbiologia , Distribuição Tecidual , DesmameRESUMO
Conventional genomic DNA extraction protocols need expensive and hazardous reagents for decontamination of phenolic compounds from the extracts and are only suited for certain types of tissue. We developed a simple, time-saving and cost-efficient method for genomic DNA extraction from various types of organisms, using relatively innocuous reagents. The protocol employs a single purification step to remove contaminating compounds, using a silica column and a non-hazardous buffer, and a chaotropic-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from cell lysates. We used this system to extract genomic DNA from different tissues of various organisms, including algae (Dunaliella salina), human peripheral blood, mouse liver, Escherichia coli, and Chinese hamster ovary cells. Mean DNA yields were 20-30 µg/cm(3) from fresh tissues (comparable to yields given by commercial extraction kits), and the 260/280 nm absorbance ratio was 1.8-2.0, demonstrating a good degree of purity. The extracted DNA was successfully used in PCR, restriction enzyme digestion and for recombinant selection studies.