RESUMEN
Vitamin D (25(OH)D3) is an essential nutrient that plays a role in the immune system. Serum 25(OH)D3 is found to be associated with asthma. However, the role of vitamin D in obese asthma remains unclear. Therefore, we investigated the association between vitamin D levels and asthma outcomes in a murine model of obese asthma. We also evaluated NLRP3 inflammasome activity in the pathogenesis of obese asthma. We divided 20 male Balb/c mice (3-4 weeks old) into 4 groups: normal control, asthma, obese, and obese asthma and developed an obese asthma mouse model. Airway hyperreactivity, cytokine concentrations, 25(OH)D3 levels, NLRP3 mRNA and IL-1β mRNA expressions were measured. Lung histology and bronchoalveolar lavage fluid (BALF) cell count were also determined. Obese asthma mice showed a significant increase in airway hyper-responsiveness, airway inflammation, pro-inflammatory cytokine levels and NLRP3 mRNA, IL-1β mRNA expression. Both asthma and obese groups had lower 25(OH)D3 levels. Vitamin D levels in obese asthma were the lowest among all groups. Vitamin D levels correlated negatively with body weight, lung resistance levels at 25 mg/mL of methacholine, total inflammatory cells, and IL-1β and IL-17 concentrations in BALF. These data demonstrated an association between serum vitamin D levels and outcomes of obese asthma, and indicated that NLRP3 inflammasome may play a role in this disorder.
Asunto(s)
Animales , Masculino , Asma/fisiopatología , Asma/metabolismo , Colecalciferol/sangre , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Obesidad/fisiopatología , Obesidad/metabolismo , Asma/patología , Factores de Tiempo , Peso Corporal , Ensayo de Inmunoadsorción Enzimática , Líquido del Lavado Bronquioalveolar , Citocinas/análisis , Citocinas/metabolismo , Modelos Animales de EnfermedadRESUMEN
Vitamin D (25(OH)D3) is an essential nutrient that plays a role in the immune system. Serum 25(OH)D3 is found to be associated with asthma. However, the role of vitamin D in obese asthma remains unclear. Therefore, we investigated the association between vitamin D levels and asthma outcomes in a murine model of obese asthma. We also evaluated NLRP3 inflammasome activity in the pathogenesis of obese asthma. We divided 20 male Balb/c mice (3-4 weeks old) into 4 groups: normal control, asthma, obese, and obese asthma and developed an obese asthma mouse model. Airway hyperreactivity, cytokine concentrations, 25(OH)D3 levels, NLRP3 mRNA and IL-1ß mRNA expressions were measured. Lung histology and bronchoalveolar lavage fluid (BALF) cell count were also determined. Obese asthma mice showed a significant increase in airway hyper-responsiveness, airway inflammation, pro-inflammatory cytokine levels and NLRP3 mRNA, IL-1ß mRNA expression. Both asthma and obese groups had lower 25(OH)D3 levels. Vitamin D levels in obese asthma were the lowest among all groups. Vitamin D levels correlated negatively with body weight, lung resistance levels at 25 mg/mL of methacholine, total inflammatory cells, and IL-1ß and IL-17 concentrations in BALF. These data demonstrated an association between serum vitamin D levels and outcomes of obese asthma, and indicated that NLRP3 inflammasome may play a role in this disorder.
Asunto(s)
Asma/metabolismo , Asma/fisiopatología , Colecalciferol/sangre , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Obesidad/metabolismo , Obesidad/fisiopatología , Animales , Asma/patología , Peso Corporal , Líquido del Lavado Bronquioalveolar , Citocinas/análisis , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Recuento de Leucocitos , Pulmón/patología , Masculino , Ratones Endogámicos BALB C , Obesidad/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de TiempoRESUMEN
We investigated the association between the interleukin 4 gene (IL-4) -590C>T polymorphism and forced expiratory volume in one second (FEV1) values, immunoglobulin E (IgE) levels, and susceptibility to asthma in Uighur children. IL-4 -590C>T frequencies were analyzed in 38 bronchial asthmatic patients and 35 non-asthmatic controls. Polymerase chain reaction and direct sequencing were applied to determine the residue at position -590 of IL-4. Total serum IgE levels were detected by enzyme-linked immunosorbent assay, while lung function was examined by professionals. There were significant differences in the distribution of IL-4 -590C>T genotypes and alleles between patient and control groups (genotypes: chi-square = 11.476, P < 0.05; alleles: chi-square = 14.572, P < 0.05). Frequencies of CC, CT, and TT genotypes were 21, 29, and 50% among patients, and 49, 37, and 14% among controls, respectively, indicating that the T allele was significantly more frequent in the asthma group than in the control group. Total serum IgE levels were significantly higher (P < 0.05) and FEV1 values were significantly lower (F = 13.294, P < 0.05) in patients than in control subjects of the same genotype. In conclusion, the IL-4 -590C>T polymorphism is related to bronchial asthma in Uighur children, and the T allele may constitute a susceptibility factor in this group. Furthermore, this genetic variant can result in raised IgE levels and decreased FEV1 values, suggesting that both factors are associated with bronchial asthma in Uighur children.
Asunto(s)
Asma/genética , Interleucina-4/genética , Adolescente , Alelos , Pueblo Asiatico/genética , Niño , Preescolar , China , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Humanos , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
OBJECTIVE: To analyze the clinical characteristics, outcome and diagnosis of two cases of imported children Zika virus infection in China. METHOD: A retrospective analysis was performed on clinical characteristics, treatment and outcome of two cases of imported children with Zika virus infection in February 2016 in Enping People's Hospital of Guangdong. RESULT: Two cases of children with imported Zika virus infection resided in an affected area of Venezuela, 8-year-old girl and her 6 year-old brother. The main findings on physical examination included the following manifestations: fever, rash, and conjunctivitis. The rash was first limited to the abdomen, but extended to the torso, neck and face, and faded after 3-4 d. The total number of white blood cells was not high and liver function was normal. The diagnosis of two cases of Zika virus infection was confirmed by the expert group of Guangdong Provincial Center for Disease Control and Prevention, according to the epidemiological history, clinical manifestations and Zika virus nucleic acid detection results.Treatment of Zika virus infection involves supportive care. Two Zika virus infection children had a relatively benign outcome. CONCLUSION: At present, Zika virus infection in children is an imported disease in China. No specific therapy is available for this disease. Information on long-term outcomes among infants and children with Zika virus disease is limited, routine pediatric care is advised for these infants and children.
Asunto(s)
Infección por el Virus Zika/diagnóstico , Virus Zika/aislamiento & purificación , Niño , China , Exantema , Femenino , Fiebre , Humanos , Masculino , Estudios Retrospectivos , VenezuelaRESUMEN
Desmoglein 4 (DSG4) has an important role in the development of wool traits in domestic animals. The full-length DSG4 gene, which contains 3918 bp, a complete open-reading-frame, and encodes a 1040-amino acid protein, was amplified from Liaoning cashmere goat. The sequence was compared with that of DSG4 from other animals and the results show that the DSG4 coding region is consistent with interspecies conservation. Thirteen single-nucleotide polymorphisms (SNPs) were identified in a highly variable region of DSG4, and one SNP (M-1, G>T) was significantly correlated with white and black coat color in goat. Haplotype distribution of the highly variable region of DSG4 was assessed in 179 individuals from seven goat breeds to investigate its association with coat color and its differentiation among populations. However, the lack of a signature result indicates DGS4 haplotypes related with the color of goat coat.
Asunto(s)
Desmogleínas/genética , Cabras/metabolismo , Color del Cabello/genética , Polimorfismo de Nucleótido Simple , Animales , Cabras/genética , Haplotipos , Filogenia , Análisis de Secuencia de ARNRESUMEN
Dilated cardiomyopathy (DCM) is a myocardial disease with a high mortality rate. Approximately 40 genes have been found to be associated with DCM to date. Non-familial DCM can also be caused by gene mutations, suggesting that genetic factors were involved in the pathogenesis of DCM; therefore genetic testing is beneficial for the early diagnosis of DCM, which can facilitate the implementation of preventive measures by and within patient's families. Here, we investigated the underlying genetic mutations involved in the cause of patients with DCM. This prospective study included 240 patients with idiopathic DCM and 240 healthy volunteers. Subject clinical data were collected and polymerase chain reaction amplification was carried out on subject DNA for three candidate genes tropomyosin (TPM1), cardiac troponin T type-2 (TNNT2), and nuclear lamina protein A/C. Single nucleotide polymorphism (SNP) loci were detected in the TPM1 (rs1071646) and TNNT2 (rs3729547) genes, respectively. The genotype distributions and allele frequencies were found to satisfy Hardy-Weinberg equilibrium, which indicated that the group was representative. Statistically significant differences were found between the variant frequencies in the two SNP loci between the Kazakh patients with idiopathic DCM (IDCM) and healthy volunteers. A significant difference in the genotype distributions (P = 0.000) and allele frequencies (P = 0.000) of SNP rs1071646, and another significant difference in the genotype distributions (P = 0.000) and allele frequencies (P = 0.039) of SNP rs3729547 between Kazakhs with IDCM and Kazakh controls. These results suggest that the TPM1 (rs1071646) and TNNT2 (rs3729547) gene variants might represent risk factors for patients with DCM in the Kazakh population.
Asunto(s)
Cardiomiopatía Dilatada/genética , Tropomiosina/genética , Troponina T/genética , Adulto , Secuencia de Bases , Estudios de Casos y Controles , Femenino , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Mutación , Polimorfismo de Nucleótido Simple , Estudios Prospectivos , Sarcómeros/genética , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Vectores Genéticos/genética , Regiones de Fijación a la Matriz , Factor de Crecimiento Nervioso/genética , Animales , Células CHO , Cricetulus , Orden Génico , Plásmidos/genéticaRESUMEN
Recent data have indicated that inflammation may have an important correlation with obstructive sleep apnea (OSA). Studies have indicated a relationship between OSA and TNF-α gene polymorphisms. Zinc finger protein 36 (ZFP36) regulates TNF-α mRNAs. However, ZFP36 gene polymorphisms have not been investigated in OSA. Therefore, we conducted the present case-control study to assess whether variances in ZFP36 gene polymorphisms account for differences in TNF-α levels in patients with moderate-to-severe OSA. This case-control study aims to investigate the relationship between genetic variations in the ZFP36 gene and moderate-to-severe OSA. Three common single nucleotide polymorphisms of the ZFP36 gene (rs251864, rs3746083, and rs17879933) were evaluated in a group of patients with moderate-to-severe OSA (N = 408) and in a control group (N = 394) by using TaqMan polymerase chain reaction analysis. The moderate-to-severe OSA group and the control group exhibited significant differences in the distributions of rs251864 and rs17879933 genotypes and alleles (P < 0.05). TNF-α levels were significantly different not only among the three rs251864 genotypes but also between the II genotype and the DD + ID genotypes of rs17879933. However, no significant differences in sleep apnea parameters in the three ZFP36 gene polymorphisms were observed. Logistic regression analyses demonstrated that TNF-α and the three ZFP36 gene polymorphisms were not independently associated with OSA. ZFP36 might be involved in TNF-α regulation. However, ZFP36 gene variants were not independent risk factors for moderate-to-severe OSA.
Asunto(s)
Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , Apnea Obstructiva del Sueño/genética , Tristetraprolina/genética , Factor de Necrosis Tumoral alfa/genética , Adulto , Alelos , Estudios de Casos y Controles , Femenino , Regulación de la Expresión Génica , Frecuencia de los Genes , Genotipo , Humanos , Inflamación , Modelos Logísticos , Masculino , Persona de Mediana Edad , Polisomnografía , ARN Mensajero/metabolismo , Factores de Riesgo , Índice de Severidad de la Enfermedad , Transducción de Señal , Apnea Obstructiva del Sueño/metabolismo , Apnea Obstructiva del Sueño/patología , Tristetraprolina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
DRA encodes the alpha chain of the DR heterodimer, is closely linked to DRB and is considered almost monomorphic in major histocompatibility complex region. In this study, we identified the exon 2 of DRA to evaluate the immunogenetic diversity of Chinese south indigenous goat. Two single nucleotide polymorphisms in an untranslated region and one synonymous substitution in coding region were identified. These data suggest that high immunodiversity in native Chinese population.
Asunto(s)
Cabras/genética , Cadenas alfa de HLA-DR/genética , Polimorfismo Genético , Animales , Animales Domésticos , China , Exones , Cabras/clasificación , Cabras/inmunología , Cadenas alfa de HLA-DR/inmunología , Intrones , Sistemas de Lectura Abierta , Regiones no TraducidasRESUMEN
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.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/genética , ADN Intergénico/genética , Vectores Genéticos/química , Regiones de Fijación a la Matriz , Plásmidos/química , Animales , Células CHO , Línea Celular Transformada , Cloranfenicol O-Acetiltransferasa/metabolismo , Cricetulus , ADN Intergénico/metabolismo , Dosificación de Gen , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos/metabolismo , Plásmidos/metabolismo , TransgenesRESUMEN
The Yangtze River Delta white goat is a goat breed that can produce high quality brush hair (Type III hair) around the world. This study aimed to compare Type III hair and non-Type III hair goat skin tissues using differentially expressed proteins based on 2-dimensional gel electrophoresis technology. The differentially expressed protein spots were analyzed using the PDquest 8.0 software. Ten protein spots were detected as positive for mass spectrometric analysis based on a threshold of 2-fold change. Through matching based on Ultraflex III TOF/TOF and MASCOT database, four differentially expressed proteins were identified. Fibrinogen beta chain isoform 1 and ATP synthase beta subunit were upregulated in Type III hair, while succinyl-CoA:3-ketoacid-coenzyme A transferase 1-mitochondrial-like and actin-cytoplasmic 1 were upregulated in non-Type III hair. The 4 proteins play important roles in different aspects of hair follicle development. These findings could pave a good foundation for explaining the mechanism of forming Type III hair.
Asunto(s)
Cabras/metabolismo , Proteínas/metabolismo , Proteómica/métodos , Piel/metabolismo , Secuencia de Aminoácidos , Animales , Electroforesis en Gel Bidimensional , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Mapeo Peptídico , RíosRESUMEN
The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS)1, L2: (GGGGS)2, and L3:(GGGGS)3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.
Asunto(s)
Proteínas Anfibias/genética , Antineoplásicos/farmacología , Clonación Molecular/métodos , Pichia/genética , Proteínas Recombinantes de Fusión/genética , Ribonucleasas/genética , Proteínas Anfibias/biosíntesis , Proteínas Anfibias/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Expresión Génica , Humanos , Concentración 50 Inhibidora , Extracción Líquido-Líquido , Pichia/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Ingeniería de Proteínas , Señales de Clasificación de Proteína , Rana pipiens/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/farmacología , Rodaminas/química , Ribonucleasas/biosíntesis , Ribonucleasas/farmacología , Albúmina Sérica/biosíntesis , Albúmina Sérica/genética , Relación Estructura-Actividad , Transformación GenéticaRESUMEN
Gene expression data acquired at different times after traumatic brain injury (TBI) were analyzed to identify differentially expressed genes (DEGs). Interaction network analysis and functional enrichment analysis were performed to extract valuable information, which may benefit diagnosis and treatment of TBI. Microarray data were downloaded from Gene Expression Omnibus and pre-treated with MATLAB. DEGs were screened out with the SAM method. Interaction networks of the DEGs were established, followed by module analysis and functional enrichment analysis to obtain insight into the molecular mechanisms. A total of 39 samples at six time points (30 min, 4, 8, 24 , 72 h, and 21 days) were analyzed and generated 377 DEGs. Eight modules were identified from the networks and network ontology analysis revealed that cell surface receptor-linked signaling pathway, response to wounding and signaling pathway were significantly overrepresented. Altered risk genes and modules in TBI were uncovered through comparing the gene expression data acquired at various time points. These genes or modules could be potential biomarkers for diagnosis and treatment of TBI.
Asunto(s)
Lesiones Encefálicas/genética , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Análisis de Secuencia por Matrices de Oligonucleótidos , Animales , Regulación hacia Abajo/genética , Ratones , Regulación hacia Arriba/genéticaRESUMEN
Numerous studies have evaluated the association between the X-ray repair cross-complementing group 3 (XRCC3) T241M polymorphism and lung cancer risk; however, the actual association is controversial. We examined whether the T241M polymorphism confers a lung cancer risk in China. We searched the PubMed, Google Scholar, and China National Knowledge Infrastructure databases to identify studies that examined the association between the XRCC3 T241M polymorphism and the risk of lung cancer. We estimated the pooled odds ratio with its 95% confidence interval to assess this association. A total of 3977 patients with lung cancer and 3761 controls from 8 comparative studies were included in this meta-analysis. The meta-analysis results revealed no significant association between the XRCC3 T241M polymorphism and lung cancer risk. In the subgroup analysis, 6 studies with sample sizes over 500 found that the T241M polymorphism had no association with lung cancer. The XRCC3 T241M polymorphism may not be a risk factor for lung cancer. However, larger studies involving a stratified case-control population and biological characterization are needed to validate this finding.
Asunto(s)
Pueblo Asiatico/genética , Proteínas de Unión al ADN/genética , Etnicidad/genética , Predisposición Genética a la Enfermedad , Neoplasias Pulmonares/genética , Polimorfismo de Nucleótido Simple/genética , China , Humanos , Oportunidad Relativa , Sesgo de Publicación , Factores de RiesgoRESUMEN
This study aimed to investigate the value of magnetic resonance spectroscopy (MRS) imaging in assessing nasopharyngeal carcinoma radiotherapy during the early delayed reaction period. Eighty cases of nasopharyngeal cancer treated with radiotherapy within the same period underwent MRS imaging before or after radiotherapy. Of the 80 cases, 47 underwent MRS imaging on the 3rd, 4th, 6th, and 12th months after radiotherapy. The trends of the primary metabolite concentration at different time points were monitored and compared with the corresponding data after radiotherapy. Repeated measures analysis of variance was performed. At the end of radiotherapy, the N-acetyl aspartate (NAA)/creatine (Cr), choline (Cho)/Cr, and NAA/Cho ratios were reduced to the lowest levels after 3 months. However, increasing trends were observed from the 4th to the 12th month. On the 12th month, stable levels were reached with statistically significant differences (F = 316.02, 53.84, 286.68; P < 0.01). MRS reflected the radiation injury-repair process in the brain of a nasopharyngeal cancer patient during early delayed reaction. This non-invasive monitoring of changes in brain tissue metabolite concentrations provides valuable information for prognosis.
Asunto(s)
Lesiones Encefálicas/diagnóstico , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/métodos , Neoplasias Nasofaríngeas/radioterapia , Traumatismos por Radiación/diagnóstico , Adulto , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Lesiones Encefálicas/etiología , Lesiones Encefálicas/metabolismo , Colina/metabolismo , Creatina/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Traumatismos por Radiación/etiología , Traumatismos por Radiación/metabolismo , Radioterapia/efectos adversos , Radioterapia/métodos , Factores de TiempoRESUMEN
ATP-binding cassette super family (ABC) proteins are considered key to oncology and pharmacology studies. We examined the effect of benzene on ABC pump protein levels in C57BL/6 mouse bone marrow mononuclear cells. After a 2-week gavage (200 mg/kg, 5 days per week), the number of peripheral leukocytes, lymphocytes and basophils dropped significantly; there was also a significant decrease in MDR1 and MRP1 gene expression. A significant reduction in expression of P-gp was found; however, there was no significant decrease in the expression of MRP1 and NF-κB p65. We conclude that regulation of membrane efflux transport protein could be a factor in benzene hematotoxicity.
Asunto(s)
Benceno/toxicidad , Monocitos/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Basófilos/efectos de los fármacos , Basófilos/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ratones , Monocitos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Factor de Transcripción ReIA/genéticaRESUMEN
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.