RESUMEN
Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase (AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line (DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus. The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Linfocitos B/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/enzimología , Línea Celular Tumoral , Pollos , Chlorocebus aethiops , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Doxiciclina/farmacología , Expresión Génica/efectos de los fármacos , Hipermutación Somática de Inmunoglobulina/efectos de los fármacos , Hipermutación Somática de Inmunoglobulina/genética , Células Vero , Proteínas no Estructurales Virales/inmunología , Virus Zika/inmunología , Infección por el Virus Zika/virologíaRESUMEN
We previously developed the in vitro method to generate monoclonal antibodies (mAbs) from libraries constructed with chicken B-cell line DT40 (referred to as the "ADLib system"). As the wild-type DT40 cells express immunoglobulin M (IgM), the original ADLib system provides monoclonal antibodies in chicken IgM format. For the therapeutic, diagnostic, and research purposes, the Fc regions of IgMs should be exchanged to other classes and species, for example human or murine IgG. However, the Fc engineering by conventional bioengineering process is laborious and takes plenty of time. Here, we developed a method to enable the seamless replacement of the Fc regions of antibodies generated by the ADLib system, using recombination-mediated cassette exchange (RMCE). In this system, two Cre recombinase recognition sites were inserted into the IgM's Fc region of the DT40 genome, allowing the exchange of the Fc region to the sequences of interest by co-transfection of a donor sequence and a Cre recombinase expression vector. We describe the detailed protocol of the technology: how to construct the RMCE host strains, select mAbs by the ADLib system, and exchange their Fc regions to generate chimeric mAbs.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión , Animales , Anticuerpos Monoclonales/química , Formación de Anticuerpos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biblioteca de Genes , Recombinación Homóloga , Humanos , Inmunoensayo , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Recombinación GenéticaRESUMEN
Infectious bursal disease virus (IBDV) is an important immunosuppressive virus in chickens. Surface immunoglobulin M (sIgM)-bearing B lymphocytes act as the major targets of IBDV in the bursa of Fabricius, and sIgM may function as one of the membrane binding sites responsible for IBDV infection. Recently, using the virus overlay protein binding assay, the chicken λ light chain of sIgM was identified to specifically interact with IBDV in a virulence-independent manner in vitro. To further investigate sIgM λ light chain-mediated IBDV binding and infection in pre-B cells, the cell line DT40, which is susceptible to both pathogenic and attenuated IBDV, was used. Based on the RNA interference strategy, the DT40 cell line whose λ light chain of sIgM was stably knocked down, herein termed DT40LKD, was generated by the genomic integration of a specific small hairpin RNA and a green fluorescence protein co-expression construct. Flow cytometry analysis indicated that the binding of IBDV to DT40LKD cells was significantly reduced due to the loss of sIgM λ light chain. In particular, reduced viral replication was observed in IBDV-incubated DT40LKD cells, and no viral release into cell culture medium was detected by the IBDV rapid diagnostic strips. In addition, the rescue of sIgM λ light chain expression restored viral binding and replication in DT40LKD cells. These results show that sIgM λ light chain appears to be beneficial for IBDV attachment and infection, suggesting that sIgM acts as a binding site involved in IBDV infection.
Asunto(s)
Linfocitos B/virología , Cadenas Ligeras de Inmunoglobulina/metabolismo , Inmunoglobulina M/metabolismo , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Proteínas de la Membrana/metabolismo , Receptores Virales/metabolismo , Acoplamiento Viral , Animales , Infecciones por Birnaviridae/veterinaria , Infecciones por Birnaviridae/virología , Línea Celular , Pollos , Técnicas de Silenciamiento del Gen , Prueba de Complementación Genética , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Modelos Biológicos , Enfermedades de las Aves de Corral/virología , Interferencia de ARN , Replicación ViralRESUMEN
BACKGROUND: In the recent past, many studies have been focused on extracts of BF and multiple biologically active factors and their effects on humoral immune system in chickens and birds. However, the mechanism of those immunomodulatory peptides on the B lineage cells proliferation and antibody production in chicken is fairly unknown. DT40 cell line, an avian leucosis virus-induced chicken pre-B cell line, expresses immunoglobulin M (IgM) isotype B cell reporter in the plasma membrane. There are many evidences suggesting that DT40 cells are best characterized as a bursal stem cell line. Because of the unique characteristics of DT40 cell line, it has been widely used to observe biological processes of pre-B lymphocyte cell within living cells. METHODS: The chicken B cell line DT40 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium and cytotoxicity was studied. Also, effect of BP5 on cell proliferation and cell cycle distribution of DT40 cells was studied. Also, the effect of BP5 on sIgM mRNA expression was studied by using real-time PCR. OBJECTIVES: To investigat the effects of Bursopentin (Cys-Lys-Arg-Val-Tyr, BP5) on a chicken promyelocyte cell line DT40, assays of cell proliferation, cell cycle distribution, detection of surface immunoglobulin G (sIgM) mRNA expression and gene microarray analysis were performed. RESULTS: The results showed that BP5 displayed concentration-dependent effects on the proliferation, cell cycle, and sIgM mRNA expression in DT40 cells. And the analysis of expression profiles identified a signature set of 3022 genes (1254 up regulated genes, 1762 down regulated genes), which clearly discriminated the BP5-treated DT40 cells from control with high certainty (P≤0.02). The results of microarray analysis were confirmed by quantitative reverse transcription-polymerase chain reaction for 12 of the differentially expressed genes. CONCLUSION: Theses findings showed the immuno-activity effect of BP5 on B lymphocyte and indicated that BP5 treatment regulated eight signaling pathways, in which Toll-like signaling pathway was the most significant enrichment pathway.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Pollos/inmunología , Células Precursoras de Granulocitos/efectos de los fármacos , Inmunoglobulina M/biosíntesis , Oligopéptidos/farmacología , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero , Receptores de Antígenos de Linfocitos B/biosíntesis , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BRCA1 plays a central role in DNA repair. Although N-terminal RING and C-terminal BRCT domains are studied well, the functions of the central region of BRCA1 are poorly characterized. Here, we report a structural and functional analysis of BRCA1 alleles and functional human BRCA1 in chicken B-lymphocyte cell line DT40. The combination of "homologous recombineering" and "RT-cassette" enables modifications of chicken BRCA1 gene in Escherichia coli. Mutant BRCA1 knock-in DT40 cell lines were generated using BRCA1 mutation constructs by homologous recombination with a targeting efficiency of up to 100%. Our study demonstrated that deletion of motifs 2-9 BRCA1Δ/Δ181-1415 (Caenorhabditis elegans BRCA1 mimic) or deletion of motif 1 BRCA1Δ/Δ126-136 decreased cell viability following cisplatin treatment. Furthermore, deletion of motifs 5 and 6 BRCA1Δ/Δ525-881 within DNA-binding region, even the conserved 7-amino acid deletion BRCA1Δ/Δ872-878 within motif 6, caused a decreased cell viability upon cisplatin treatment. Surprisingly, human BRCA1 is functional in DT40 cells as indicated by DNA damage-induced Rad 51 foci formation in human BRCA1 knock-in DT40 cells. These results demonstrate that those conserved motifs within the central region are essential for DNA repair functions of BRCA1. These findings provide a valuable tool for the development of new therapeutic modalities of breast cancer linked to BRCA1.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Alelos , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Pollos , Cisplatino/farmacología , Daño del ADN/genética , Reparación del ADN , Femenino , Humanos , Linfoma de Células B , Mutación , Proteínas Nucleares/metabolismo , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
BACKGROUND AND PURPOSE: Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are intracellular Ca(2+) channels. Interactions of the commonly used antagonists of IP3Rs with IP3R subtypes are poorly understood. EXPERIMENTAL APPROACH: IP3-evoked Ca(2+) release from permeabilized DT40 cells stably expressing single subtypes of mammalian IP3R was measured using a luminal Ca(2+) indicator. The effects of commonly used antagonists on IP3-evoked Ca(2+) release and (3) H-IP3 binding were characterized. KEY RESULTS: Functional analyses showed that heparin was a competitive antagonist of all IP3R subtypes with different affinities for each (IP3R3 > IP3R1 ≥ IP3R2). This sequence did not match the affinities for heparin binding to the isolated N-terminal from each IP3R subtype. 2-aminoethoxydiphenyl borate (2-APB) and high concentrations of caffeine selectively inhibited IP3R1 without affecting IP3 binding. Neither Xestospongin C nor Xestospongin D effectively inhibited IP3-evoked Ca(2+) release via any IP3R subtype. CONCLUSIONS AND IMPLICATIONS: Heparin competes with IP3, but its access to the IP3-binding core is substantially hindered by additional IP3R residues. These interactions may contribute to its modest selectivity for IP3R3. Practicable concentrations of caffeine and 2-APB inhibit only IP3R1. Xestospongins do not appear to be effective antagonists of IP3Rs.
Asunto(s)
Calcio/metabolismo , Heparina/farmacología , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Animales , Compuestos de Boro/farmacología , Cafeína/farmacología , Línea Celular , Pollos , Compuestos Macrocíclicos/farmacología , Oxazoles/farmacologíaRESUMEN
Bisphenol A (BPA) has been found in plastic food containers, paper currencies and toys. BPA has been reported for various adverse health concerns including reproduction, development and carcinogenesis. These potential health implications have led to increasing use of alternative bisphenols such as bisphenol F and bisphenol S among many. However, little is known about the toxicity of alternative bisphenols and most of the toxicological information is limited to endocrine disrupting potentials. In this study, we evaluated cytotoxicity and the genotoxic potentials of several bisphenol compounds, and identified the mechanism of genotoxicity using a panel of mutant chicken DT40 cell lines deficient in DNA repair pathways. Several bisphenols including bisphenol AP, bisphenol M, or bisphenol P exerted genotoxic potentials that are greater than that of BPA. Generally RAD54(-/-) mutant cells were the most sensitive to all bisphenols except for bisphenol F, suggesting the induction of DNA double-strand breaks that could be rescued by homologous recombination. Genotoxic potential of bisphenols was confirmed by chromosomal aberration assay and γ-H2AX foci forming assay between wild-type and RAD54(-/-) mutant. Among the tested bisphenols, BPP at 12.5µM showed the greatest genotoxic potency, inducing chromosomal aberration and γ-H2AX foci in RAD54(-/-) mutant by 2.6 and 4.8 folds greater than those in wild-type, respectively. Our results clearly show several alternative bisphenols can cause genotoxicity that could be rescued by homologous recombination pathway, and some bisphenols induced even greater genotoxic potentials than that of BPA.