RESUMEN
Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides. Here, we describe a method for constructing complex plasmid libraries based on pooled designed oligomers with high representation and tight distributions. The procedure allows construction of plasmid libraries of >60,000 members with a 95th/5th percentile ratio of less than 3.5.
Asunto(s)
Sistemas CRISPR-Cas/genética , Clonación Molecular/métodos , Técnicas de Inactivación de Genes/métodos , Biblioteca de Genes , Plásmidos/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
Vector construction and gene cloning are ubiquitous techniques essential to all fields of biological and medical research. They are the first steps in many endeavors leading to expressing proteins to understand gene function and regulation. However, they can often be rate-limiting, particularly in multi-gene studies, due to the time and effort required to assemble gene constructs and to identify the optimal constructs for protein expression.The SureVector system was developed to address this by enabling the rapid and reliable assembly of multiple DNA modules into a recombinant plasmid containing a gene-of-interest (GOI). It harnesses the power of synthetic biology to combine DNA modules from standard parts into a customized vector that expresses proteins in bacterial, mammalian, or yeast cells. The key advantages of the innovative SureVector system include rapid custom vector generation, enhanced flexibility to assemble new vectors quickly as experimental requirements change, and the reliable and precise assembly of fully interchangeable standard DNA modules that retain their functionality. The SureVector system is the only next-generation plasmid assembly technology to guarantee assembly of multiple functional DNA modules.
Asunto(s)
Eucariontes/genética , Células Eucariotas/metabolismo , Expresión Génica/genética , Vectores Genéticos/genética , Células Procariotas/metabolismo , Proteínas/genética , Animales , Bacterias/genética , Clonación Molecular/métodos , ADN/genética , Mamíferos/genética , Plásmidos/genética , Recombinación Genética/genética , Biología Sintética/métodos , Levaduras/genéticaRESUMEN
A novel polymeric microfluidic device with an on-chip enzyme reactor has been developed for the characterization of recombinant glycoproteins. The enzyme reactor chip packed with PNGase F-modified solid support material was combined with a microfluidic glycan cleanup chip and a commercially available HPLC-chip to perform glycoprotein deglycosylation, protein removal, glycan capture, glycan LC separation, and nanoelectrospray into a time-of-flight mass spectrometry (TOF-MS) system. With this integrated chip, the combined sample preparation and sample analysis time was reduced from multiple hours to less than 10 min. A once tedious and time-consuming glycan analysis workflow is now integrated into an HPLC-chip device. Glycan profiling analysis has been achieved with as little as 100 ng of monoclonal antibody. Furthermore, a single chip was shown to retain activity and perform equivalently for over 250 replicate glycan profiles from a recombinant antibody.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Técnicas Analíticas Microfluídicas/métodos , Polisacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Enzimas Inmovilizadas/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
We describe a set of Moloney Murine Leukemia Virus (MoMLV)-based replication-defective retroviral vectors for delivery of the ecdysone-inducible system into mammalian cells. The vector pFB-ERV contains a tricistronic CMV expression cassette from which the ecdysone receptor proteins RXR and VgEcR are expressed, with the neo-resistance marker expressed as the third open reading frame (ORF). The inducible vector pCFB-EGSH contains an ecdysone-inducible expression cassette inserted between the viral LTRs in the antisense orientation relative to that for the viral promoter. Potential interference from the proviral 5' LTR is obviated due to a SIN deletion in the 3' LTR. When used together, induction ratios of over 1000-fold were achieved in NIH3T3 cells using firefly luciferase as a reporter.
Asunto(s)
Ecdisona/biosíntesis , Ecdisona/genética , Marcación de Gen/métodos , Ingeniería Genética/métodos , Riñón/metabolismo , Virus de la Leucemia Murina de Moloney/genética , Transfección/métodos , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Regulación de la Expresión Génica/genética , Humanos , Riñón/embriología , Proteínas Recombinantes/biosíntesisRESUMEN
Y box-binding protein 1 (YB-1) is a multifunctional protein that can act as a regulator of transcription and of translation. In chicken embryo fibroblasts transformed by the oncoproteins P3k (phosphatidylinositol 3-kinase) or Akt, YB-1 is transcriptionally down-regulated. Expression of YB-1 from a retroviral vector induces a strong cellular resistance to transformation by P3k or Akt but does not affect sensitivity to transformation by other oncoproteins, such as Src, Jun, or Qin. The YB-1-expressing cells assume a tightly adherent, flat phenotype, with YB-1 localized in the cytoplasm, and show a greatly reduced saturation density. Both cap-dependent and cap-independent translation is inhibited in these cells, but the activity of Akt remains unaffected, suggesting that YB-1 functions downstream of Akt. A YB-1 protein with a loss-of-function mutation in the RNA-binding motif no longer binds to the mRNA cap structure, is localized in the cell nucleus, does not induce the flat cellular phenotype, and fails to interfere with P3k- or Akt-induced oncogenic transformation. This mutant also does not inhibit cap-dependent or cap-independent translation. These results suggest that YB-1 acts like a rapamycin mimic, inhibiting translational events that are required in phosphatidylinositol 3-kinase-driven oncogenic transformation.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Factores de Transcripción , Animales , Secuencia de Bases , Sitios de Unión , División Celular , Células Cultivadas , Embrión de Pollo , ADN/genética , Expresión Génica , Factores de Transcripción NFI , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína 1 de Unión a la Caja YRESUMEN
Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.
Asunto(s)
Proteínas de Ciclo Celular , Clonación Molecular/métodos , ADN Complementario/metabolismo , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Vectores Genéticos , Retroviridae/genética , Retroviridae/metabolismo , Células 3T3/metabolismo , Animales , Secuencia de Bases , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Oncogenes/genética , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf/análisis , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas c-vav , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Mapeo RestrictivoRESUMEN
Integrin and cell adhesion molecule-regulated cellular adhesion plays an integral part in the recruitment and activation of lymphocytes. T-cell activation is a dynamic process subject to integrin-dependent and -independent regulation. Stimulation of human peripheral blood T cells by the anti-CD3 monoclonal antibody results in a rapid upregulation of integrin affinity. In conjunction with adhesion to endothelial cell-derived ligands and extracellular matrix proteins, anti-CD3 antibodies have been shown to result in significant increases in IL-2 production and T-cell proliferation. Therefore, at least two signal cascades are activated by ligation of the TCR: One results in a change in affinity of integrins for their ligands, whereas the other activates a signaling cascade that leads to gene induction. We investigated the effects of several tyrosine kinase inhibitors on human peripheral blood T-cell adhesion and adhesion-induced costimulation of IL-2 expression and secretion. These compounds did not inhibit anti-CD3-induced short-term (30 min) or long-term (18 hr) T-cell adhesion to VCAM-1, MAdCAM, or ICAM-1. When T cells were stimulated with anti-CD3 and allowed to adhere to VCAM-1, MAdCAM, or ICAM-1 in the presence of these inhibitors; IL-2 production was significantly reduced. The MEK specific inhibitor, PD98059, did not block T-cell adhesion to the various substrates, but it did block IL-2 synthesis. In addition, the tyrosine kinase inhibitors and PD98059 blocked anti-CD3-mediated stimulation of IL-2 synthesis. These data suggest that the signaling mechanism for anti-CD3-mediated integrin activation is distinct from the signaling pathway that results in adhesion-induced IL-2 synthesis via specific integrins and anti-CD3.