RESUMEN
Reproducible and stable transgene expression is an important goal in both basic research and biotechnology, with each application demanding a range of transgene expression. Problems in achieving stable transgene expression include multi-copy transgene silencing, chromosome-position effects, and loss of expression during long-term culture, induced cell quiescence, and/or cell differentiation. Previously, we described the "BAC TG-EMBED" method for copy-number dependent, chromosome position-independent expression of embedded transgenes within a BAC containing ~170 kb of the mouse Dhfr locus. Here we demonstrate wider applicability of the method by identifying a BAC and promoter combination that drives reproducible, copy-number dependent, position-independent transgene expression even after induced quiescence and/or cell differentiation into multiple cell types. Using a GAPDH BAC containing ~200 kb of the human GAPDH gene locus and a 1.2 kb human UBC promoter, we achieved stable GFP-ZeoR reporter expression in mouse NIH 3T3 cells after low-serum-induced cell cycle arrest or differentiation into adipocytes. More notably, GFP-ZeoR expression remained stable and copy-number dependent even after differentiation of mouse ESCs into several distinct lineages. These results highlight the potential use of BAC TG-EMBED as an expression platform for high-level but stable, long-term expression of transgene independent of cell proliferative or differentiated state.
Asunto(s)
Cromosomas Artificiales Bacterianos , Transfección/métodos , Transgenes , Animales , Diferenciación Celular/genética , Técnicas de Transferencia de Gen , Genes Reporteros , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Células 3T3 NIH , Regiones Promotoras Genéticas , Tetrahidrofolato Deshidrogenasa/genética , Transformación GenéticaRESUMEN
Achieving stable expression of a single transgene in mammalian cells remains challenging; even more challenging is obtaining simultaneous stable expression of multiple transgenes at reproducible, relative expression levels. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC "scaffold" containing the mouse Msh3-Dhfr locus (DHFR BAC). Here, we extend this "BAC TG-EMBED" approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01- to 5-fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes. Second, we demonstrate little variation in expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making the choice of BAC scaffolds more flexible. Third, we present a novel BAC assembly scheme, "BAC-MAGIC", for inserting multiple transgenes into BAC scaffolds, which is much more time-efficient than traditional galK-based methods. As a proof-of-principle for our improved BAC TG-EMBED toolkit, we simultaneously fluorescently labeled three nuclear compartments at reproducible, relative intensity levels in 94% of stable clones after a single transfection using a DHFR BAC scaffold containing 4 transgenes assembled with BAC-MAGIC. Our extended BAC TG-EMBED toolkit and BAC-MAGIC method provide an efficient, versatile platform for stable simultaneous expression of multiple transgenes at reproducible, relative levels.
Asunto(s)
Cromosomas Artificiales Bacterianos , Ingeniería Genética/métodos , Transgenes/genética , Animales , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Microscopía Fluorescente , Proteína 3 Homóloga de MutS/genética , Células 3T3 NIH , Regiones Promotoras Genéticas , Tetrahidrofolato Deshidrogenasa/genética , TransfecciónRESUMEN
Bacterial artificial chromosomes (BACs) were constructed containing full-length, infectious DNA of HSV-1 strains 17 and KOS. To generate BACs without altering viral genes, sequences required for selection and propagation of the BAC were placed between the U(L)37 and U(L)38 genes, and flanked by LoxP sites. The system was tested by studying multiple properties of these HSV-1 BAC constructs in vitro and in vivo following propagation in bacteria, virus reconstitution from HSV-BAC DNA in eukaryotic cells, and Cre-recombinase-mediated excision of the BAC backbone. Based on in vitro growth in mouse embryo fibroblasts and in vivo growth in mouse corneas and trigeminal ganglia, the strain KOS BAC-derived virus behaved similarly to wild-type. Small changes in neurovirulence were, however, observed. The strain 17 BAC-derived virus exhibited modest decreases in growth and virulence compared to wild-type. Modest differences were observed in reactivation from latency with both strain KOS and 17 BAC-derived viruses. In addition, the system was further validated by performing mutagenesis of the BACs by allelic exchange in E. coli. These BACs are suitable for the rapid generation of recombinant viruses for pathogenesis and other studies, but as with all mutagenesis systems, care must be taken in their construction and repair.
Asunto(s)
Proteínas de la Cápside/genética , Cromosomas Artificiales Bacterianos , Herpesvirus Humano 1/genética , Proteínas Estructurales Virales/genética , Animales , Chlorocebus aethiops , Escherichia coli/genética , Femenino , Genoma Viral , Herpesvirus Humano 1/fisiología , Ratones , Mutagénesis , Células Vero , Activación Viral , Replicación ViralAsunto(s)
Astrocitoma/patología , Neoplasias Encefálicas/patología , Ventrículos Cerebrales/patología , Anciano , Astrocitoma/diagnóstico por imagen , Neoplasias Encefálicas/diagnóstico por imagen , Ventrículos Cerebrales/diagnóstico por imagen , Femenino , Humanos , Tomografía Computarizada por Rayos XRESUMEN
Individuals with autistic spectrum disorder (ASD) display diverse deficits in social, cognitive and behavioral functioning. To date, there has been mixed findings on the profile of executive function deficits for high-functioning adults (IQ > 70) with ASD. A conceptual distinction is commonly made between "cold" and "hot" executive functions. Cold executive functions refer to mechanistic higher-order cognitive operations (e.g., working memory), whereas hot executive functions entail cognitive abilities supported by emotional awareness and social perception (e.g., social cognition). This study aimed to determine the independence of deficits in hot and cold executive functions for high-functioning adults with ASD. Forty-two adults with ASD (64% male, aged 18-66 years) and 40 age and gender matched controls were administered The Awareness of Social Inference Test (TASIT; emotion recognition and social inference), Letter Number Sequencing (working memory) and Hayling Sentence Completion Test (response initiation and suppression). Between-group analyses identified that the ASD group performed significantly worse than matched controls on all measures of cold and hot executive functions (d = 0.54 - 1.5). Hierarchical multiple regression analyses revealed that the ASD sample performed more poorly on emotion recognition and social inference tasks than matched controls after controlling for cold executive functions and employment status. The findings also indicated that the ability to recognize emotions and make social inferences was supported by working memory and response initiation and suppression processes. Overall, this study supports the distinction between hot and cold executive function impairments for adults with ASD. Moreover, it advances understanding of higher-order impairments underlying social interaction difficulties for this population which, in turn, may assist with diagnosis and inform intervention programs.
Asunto(s)
Cordoma/patología , Neoplasias de los Tejidos Blandos/patología , Adenocarcinoma/patología , Tumor Carcinoide/patología , Neoplasias del Colon/patología , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Neoplasias Primarias Secundarias/patología , Vértebras Torácicas/patologíaRESUMEN
A central question in genomic imprinting is how parental-specific DNA methylation of imprinting control regions (ICR) is established during gametogenesis and maintained after fertilization. At the imprinted Igf2/H19 locus, CTCF binding maintains the unmethylated state of the maternal ICR after the blastocyst stage. In addition, evidence from Beckwith-Wiedemann patients and cultured mouse cells suggests that two Sox-Oct binding motifs within the Igf2/H19 ICR also participate in maintaining hypomethylation of the maternal allele. We found that the Sox and octamer elements from both Sox-Oct motifs were required to drive hypomethylation of integrated transgenes in mouse embryonic carcinoma cells. Oct4 and Sox2 showed cooperative binding to the Sox-Oct motifs, and both were present at the endogenous ICR. Using a mouse with mutations in the Oct4 binding sites, we found that maternally transmitted mutant ICRs acquired partial methylation in somatic tissues, but there was little effect on imprinted expression of H19 and Igf2. A subset of mature oocytes also showed partial methylation of the mutant ICR, which suggested that the Sox-Oct motifs provide some protection from methylation during oogenesis. The Sox-Oct motifs, however, were not required for erasure of paternal methylation in primordial germ cells, which indicated that the oocyte methylation was acquired post-natally. Maternally inherited mutant ICRs were unmethylated in blastocysts, which suggested that at least a portion of the methylation in somatic tissues occurred after implantation. These findings provide evidence that Sox-Oct motifs contribute to ICR hypomethylation in post-implantation embryos and maturing oocytes and link imprinted DNA methylation with key stem cell/germline transcription factors.