RESUMEN
The global adoption of vaccines to combat disease is hampered by the high cost of vaccine manufacturing. The work described herein follows two previous publications (van der Sanden et al., 2016; Wu et al., 2017) that report a strategy to enhance poliovirus and rotavirus vaccine production through genetic modification of the Vero cell lines used in large-scale vaccine manufacturing. CRISPR/Cas9 gene editing tools were used to knockout Vero target genes previously shown to play a role in polio- and rotavirus production. Subsequently, small-scale models of current industry manufacturing systems were developed and adopted to assess the increases in polio- and rotavirus output by multiple stable knockout cell lines. Unlike previous studies, the Vero knockout cell lines failed to achieve desired target yield increases. These findings suggest that additional research will be required before implementing the genetically engineered Vero cell lines in the manufacturing process for polio- and rotavirus vaccines to be able to supply vaccines at reduced prices.
Asunto(s)
Técnicas de Cultivo Celular por Lotes , Ingeniería Genética , Células Vero , Vacunas Virales , Animales , Sistemas CRISPR-Cas , Chlorocebus aethiops , Técnicas de Inactivación de Genes , Marcación de Gen , Poliovirus/genética , Poliovirus/inmunología , Vacunas contra Poliovirus/química , Vacunas contra Poliovirus/inmunología , Rotavirus/genética , Rotavirus/inmunología , Vacunas contra Rotavirus/genética , Vacunas contra Rotavirus/inmunologíaRESUMEN
BACKGROUND: Flow cytometry, in combination with retroviral expression libraries, is a powerful tool for genetic experimentation in mammalian cells. Expression libraries are transduced into cells engineered with a fluorescent reporter. Sorting for either bright or dim cells allows enrichment for specific inhibitors that alter reporter activity. This strategy has been used to isolate peptides and RNAs that either activate or suppress defined biochemical pathways. METHODS: Several variables contribute to the enrichment process: (1) the background of the fluorescence bioassay; (2) the mean fluorescence ratio between the induced and noninduced reporter cell populations; (3) the genetic penetrance, or strength, of the inhibitor; and (4) the multiplicity of infection (MOI). An experimental and theoretical analysis, including computer modeling, of these issues in the context of a mammalian cell bioassay was undertaken. RESULTS: MOI measurements were shown to be problematic. High MOI had little effect on enrichment early in the cycling process but a significant effect at later stages. Penetrance and background were critical throughout the process. Enrichments within about twofold of the theoretical maximum were observed. CONCLUSIONS: Caution should be exercised in MOI determination because of the danger of significant underestimation. High MOI is potentially advantageous early in the selection process but hinders enrichment in the later rounds. Modeling shows that MOI, assay background and clone penetrance are the principal variables that determine the success of transdominant selections by FACS.
Asunto(s)
Separación Celular/métodos , Citometría de Flujo/métodos , Genes Reporteros , Técnicas Genéticas , Animales , Línea Celular , Humanos , Retroviridae/fisiología , Programas Informáticos , Transducción Genética , Células Tumorales CultivadasRESUMEN
Quality bioassays are central to all approaches directed at understanding or perturbing the function of proteins. One type of cell-based bioassay involves an engineered reporter whose transcriptional activity serves as a readout for upstream signals of a biochemical pathway(s) that feeds into the reporter. We describe a general strategy for creating a mammalian reporter line with attributes suitable for a high complexity, en masse transdominant genetic screen. The basic criteria required of the mammalian cells engineered with the reporter include ease of maintenance, ease of sorting by FACS, ability to be transduced by retroviruses, and high expression of transduced peptides or cDNAs. For maximal enrichment during selection, the reporter line should have a relatively homogeneous response and a high signal-to-background ratio. We use a melanoma cell line transduced with a retinoic-acid-responsive promoter coupled to a GFP reporter as a case study to demonstrate the strategy. We characterize an optimized retinoic-acid-responsive reporter clone to determine the kinetics of reporter induction and decay in the presence and absence of retinoids. Dose-response studies reveal that the reporter responds to all-trans retinoic acid with an EC50 of approximately 1 nM. The strategy described is general and may be applied to create other reporter lines that respond to a specific stimulus.
Asunto(s)
Genes Dominantes , Genes Reporteros , Selección Genética , Secuencia de Bases , Separación Celular , ADN , Cartilla de ADN , Citometría de Flujo , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Melanoma/genética , Melanoma/patología , Retroviridae/genética , Transducción Genética , Células Tumorales CultivadasRESUMEN
The compound eye of Drosophila is a reiterated pattern of 800 unit eyes known as ommatidia. In each ommatidium there are eight photoreceptor neurons (R1-R8) and an invariant number of accessory cells organized in a precise manner. In the developing eye, specification of cell fates is triggered by sequential inductive events mediated by cell-cell interactions. The R8 photoreceptor neuron is the first cell to differentiate and is thought to play a central role in the recruitment of the remaining photoreceptor cells. Our previous work demonstrated that mutations in the retina aberrant in pattern (rap) locus lead to abnormal pattern formation in the compound eye. Genetic mosaic experiments demonstrated that for normal retinal patterning to occur, rap gene function is required only in the photoreceptor cell R8. In this study we analyzed the R cell composition of developing as well as the adult eyes of rap mutants employing a variety of R cell specific markers. We show that in rap mutants, although some of the R8-specific markers show normal expression patterns, other aspects of the R8 cell differentiation are abnormal. In addition, the cells R1, R6, and R7 fail to differentiate properly in rap mutants. These results suggest that the rap gene encodes an R8-specific function that plays a role in the determination of the photoreceptor cells R1, R6, and R7.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Drosophila , Drosophila/embriología , Glicoproteínas/genética , Células Fotorreceptoras de Invertebrados/embriología , Receptores de Péptidos , Factores de Transcripción , Animales , Comunicación Celular/fisiología , Muerte Celular/genética , Diferenciación Celular/fisiología , Cobalto , Elementos de Facilitación Genéticos/genética , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio , Hormonas de Insectos/genética , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Operón Lac , Ligandos , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Microscopía Electrónica , Chaperonas Moleculares/genética , Mutación/fisiología , Neuronas Aferentes/citología , Neuronas Aferentes/enzimología , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/ultraestructura , Proteínas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Recombinantes/genética , Retina/citología , Retina/embriología , Retina/ultraestructura , Opsinas de Bastones/genética , Coloración y Etiquetado , Transcripción Genética/fisiologíaRESUMEN
Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.
Asunto(s)
Adenosina Trifosfatasas/aislamiento & purificación , Compuestos de Dansilo , Mucosa Gástrica/enzimología , Glicoproteínas/aislamiento & purificación , Animales , Western Blotting , Cromatografía de Afinidad , Detergentes , Electroforesis en Gel de Poliacrilamida , Glicosilación , ATPasa Intercambiadora de Hidrógeno-Potásio , Hidrazinas , Técnicas de Inmunoadsorción , Lectinas , Microsomas/enzimología , Peso Molecular , Octoxinol , Péptido Hidrolasas/metabolismo , Polietilenglicoles , Compuestos de Amonio Cuaternario , Coloración y Etiquetado , PorcinosRESUMEN
In the compound eye of Drosophila, cell-cell interactions are thought to play an important role in the determination of neuronal cell fate and pattern morphogenesis. Recent work on the bride of sevenless (boss) gene has demonstrated an inductive role for photoreceptor R8 in the differentiation of photoreceptor R7. These studies have shown that while R8 differentiates early in the scheme of ommatidial assembly, it continues to play an active role in subsequent patterning events. We describe studies on a new genetic locus rap (retina aberrant in pattern), whose functions are critical for normal pattern formation in the developing eye. Mutations in the rap gene perturb the early stages of pattern formation and lead to a variable number of photoreceptor cells (R cells) in each ommatidium. Experiments with a temperature-sensitive allele have shown that rap gene function is required during the period of development when pattern formation occurs. In addition, a somatic mosaic analysis of rap has shown that its function is required only in photoreceptor cell R8 for normal ommatidial patterning. These studies suggest an important role for rap in the initial events leading to pattern formation and are consistent with R8 playing a central role in directing ommatidial pattern formation.