Rational design and construction of multi-copy biomanufacturing islands in mammalian cells

Cell line development is a critical step in the establishment of a biopharmaceutical manufacturing process. Current protocols rely on random transgene integration and amplification. Due to considerable variability in transgene integration profiles, this workflow results in laborious screening campaigns before stable producers can be identified. Alternative approaches for transgene dosage increase and integration are therefore highly desirable. In this study, we present a novel strategy for the rapid design, construction, and genomic integration of engineered multiple-copy gene constructs consisting of up to 10 gene expression cassettes. Key to this strategy is the diversification, at the sequence level, of the individual gene cassettes without altering their protein products. We show a computational workflow for coding and regulatory sequence diversification and optimization followed by experimental assembly of up to nine gene copies and a sentinel reporter on a contiguous scaffold. Transient transfections in CHO cells indicates that protein expression increases with the gene copy number on the scaffold. Further, we stably integrate these cassettes into a pre-validated genomic locus. Altogether, our findings point to the feasibility of engineering a fully mapped multi-copy recombinant protein ‘production island’ in a mammalian cell line with greatly reduced screening effort, improved stability, and predictable product titers.     

Far-upstream elements are dispensable for tissue-specific proenkephalin expression using a Cre-mediated knock-in strategy

Several cis-regulatory DNA elements are present in the 5' upstream regulatory region of the enkephalin gene (ENK) promoter. To determine their role in conferring organ-specificity of ENK expression in mice and to circumvent the position effects from random gene insertion that are known to often frustrate such analysis in transgenic mice, we used a Cre-mediated gene knock-in strategy to target reporter constructs to a "safe haven" loxP-tagged locus in the hypoxanthine phosphoribosyltransferase (HPRT) gene. Here we report reliable and reproducible reporter gene expression under the control of the 5' upstream regulatory region of the mouse ENK gene in gene-modified mice using this Cre-mediated knock-in strategy. Comparison of two 5'ENK regulatory regions (one with and the other without known cis-regulatory DNA elements) in the resulting adult mice showed that conserved far-upstream cis-regulatory DNA elements are dispensable for correct organ-specific gene expression. Thus the proximal 1.4 kb of the murine ENK promoter region is sufficient for organ-specificity of ENK gene expression when targeted to a safe-haven genomic locus. These results suggest that conservation of the far-upstream DNA elements serves more subtle roles, such as the developmental or cell-specific expression of the ENK gene.     

Enhancer detection in the zebrafish using pseudotyped murine retroviruses

Vectors based on murine retroviruses are among the most efficient means to insert reporter constructs into the context of a vertebrate chromosome with the aim to visualize cis-regulatory information available to a basal promoter at the site of insertion. In combination with using the zebrafish embryo as a readout for the activity of regulatory elements, enhancer detection becomes a powerful technique for gene discovery and for the mapping of the extent of regulatory domains in a vertebrate genome. Our laboratory has performed the only large-scale enhancer detection screen to date in any vertebrate and we describe in this paper the methods we developed to generate viral particles, to insert reporter constructs into the zebrafish germ line, the screening of detection events in heterozygous F1 embryos, and the isolation of genomic sequence flanking the inserted vector for the purpose of genomic mapping. Given sufficient scale, the technology described here can be used to obtain cis-regulatory information across the entire zebrafish genome for any given basal promoter.     

Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.     

Detection of Deregulated Modules Using Deregulatory Linked Path

The identification of deregulated modules (such as induced by oncogenes) is a crucial step for exploring the pathogenic process of complex diseases. Most of the existing methods focus on deregulation of genes rather than the links of the path among them. In this study, we emphasize on the detection of deregulated links, and develop a novel and effective regulatory path-based approach in finding deregulated modules. Observing that a regulatory pathway between two genes might involve in multiple rather than a single path, we identify condition-specific core regulatory path (CCRP) to detect the significant deregulation of regulatory links. Using time-series gene expression, we define the regulatory strength within each gene pair based on statistical dependence analysis. The CCRPs in regulatory networks can then be identified using the shortest path algorithm. Finally, we derive the deregulated modules by integrating the differential edges (as deregulated links) of the CCRPs between the case and the control group. To demonstrate the effectiveness of our approach, we apply the method to expression data associated with different states of Human Epidermal Growth Factor Receptor 2 (HER2). The experimental results show that the genes as well as the links in the deregulated modules are significantly enriched in multiple KEGG pathways and GO biological processes, most of which can be validated to suffer from impact of this oncogene based on previous studies. Additionally, we find the regulatory mechanism associated with the crucial gene SNAI1 significantly deregulated resulting from the activation of HER2. Hence, our method provides not only a strategy for detecting the deregulated links in regulatory networks, but also a way to identify concerning deregulated modules, thus contributing to the target selection of edgetic drugs.     

Developmental effector gene regulation: Multiplexed strategies for functional analysis

The staggering complexity of the genome controls for developmental processes is revealed through massively parallel cis-regulatory analysis using new methods of perturbation and readout. The choice of combinations of these new methods is tailored to the system, question and resources at hand. Our focus is on issues that include the necessity or sufficiency of given cis-regulatory modules, cis-regulatory function in the normal spatial genomic context, and easily accessible high throughput and multiplexed analysis methods. In the sea urchin embryonic model, recombineered BACs offer new opportunities for consecutive modes of cis-regulatory analyses that answer these requirements, as we here demonstrate on a diverse suite of previously unstudied sea urchin effector genes expressed in skeletogenic cells. Positively active cis-regulatory modules were located in single Nanostring experiments per BAC containing the gene of interest, by application of our previously reported “barcode” tag vectors of which>?100 can be analyzed at one time. Computational analysis of DNA sequences that drive expression, based on the known skeletogenic regulatory state, then permitted effective identification of functional target site clusters. Deletion of these sub-regions from the parent BACs revealed module necessity, as simultaneous tests of the same regions in short constructs revealed sufficiency. Predicted functional inputs were then confirmed by site mutations, all generated and tested in multiplex formats. There emerged the simple conclusion that each effector gene utilizes a small subset of inputs from the skeletogenic GRN. These inputs may function to only adjust expression levels or in some cases necessary for expression. Since we know the GRN architecture upstream of the effector genes, we could then conceptually isolate and compare the wiring of the effector gene driver sub-circuits and identify the inputs whose removal abolish expression.     

Graph-based identification of cancer signaling pathways from published gene expression signatures using PubLiME

Gene expression technology has become a routine application in many laboratories and has provided large amounts of gene expression signatures that have been identified in a variety of cancer types. Interpretation of gene expression signatures would profit from the availability of a procedure capable of assigning differentially regulated genes or entire gene signatures to defined cancer signaling pathways. Here we describe a graph-based approach that identifies cancer signaling pathways from published gene expression signatures. Published gene expression signatures are collected in a database (PubLiME: Published Lists of Microarray Experiments) enabled for cross-platform gene annotation. Significant co-occurrence modules composed of up to 10 genes in different gene expression signatures are identified. Significantly co-occurring genes are linked by an edge in an undirected graph. Edge-betweenness and k-clique clustering combined with graph modularity as a quality measure are used to identify communities in the resulting graph. The identified communities consist of cell cycle, apoptosis, phosphorylation cascade, extra cellular matrix, interferon and immune response regulators as well as communities of unknown function. The genes constituting different communities are characterized by common genomic features and strongly enriched cis-regulatory modules in their upstream regulatory regions that are consistent with pathway assignment of those genes.     

Evolution acts on enhancer organization to fine-tune gradient threshold readouts

The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.     

Splinkerette PCR for Mapping Transposable Elements in Drosophila

Transposable elements (such as the P-element and piggyBac) have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR). Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR) method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1) map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2) map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.     

Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs

We evaluate here the use of real-time quantitative PCR (q-PCR) as a method for screening for homologous recombinants generated in mammalian cells from either conventional gene-targeting constructs or whole BAC-based constructs. Using gene-targeted events at different loci, we show that q-PCR is a highly sensitive and accurate method for screening for conventional gene targeting that can reduce the number of clones requiring follow-up screening by Southern blotting. We further compared q-PCR to fluorescent in situ hybridization (FISH) for the detection of gene-targeting events using full-length BAC-based constructs designed to introduce mutations either into one gene or simultaneously into two adjacent genes. We find that although BAC-based constructs appeared to have high rates of homologous recombination when evaluated by FISH, screening by FISH was prone to false positives that were detected by q-PCR. Our results demonstrate the utility of q-PCR as a screening tool for gene targeting and further highlight potential problems with the use of whole BAC-based constructs for homologous recombination.     

Identification of brain-derived neurotrophic factor promoter regions mediating tissue-specific, axotomy-, and neuronal activity-induced expression in transgenic mice

The structure of rat brain-derived neurotrophic factor (BDNF) gene is complex; four 5' exons are linked to separate promoters and one 3' exon is encoding the BDNF protein. To analyze the relative importance of the regulatory regions in vivo, we have generated transgenic mice with six different promoter constructs of the BDNF gene fused to the chloramphenicol acetyl transferase reporter gene. High level and neuronal expression of the reporter gene, that in many respects recapitulated BDNF gene expression, was achieved by using 9 kb of genomic sequences covering the promoter regions that lie adjacent to each other in the genome (promoters I and II and promoters III and IV, respectively) and by including sequences of BDNF intron-exon splice junctions and 3' untranslated region in the constructs. The genomic regions responsible for the in vivo upregulation of BDNF expression in the axotomized sciatic nerve and in the brain after kainic acid-induced seizures and KCl-induced spreading depression were mapped. These data show that regulation of the different aspects of BDNF expression is controlled by different regions in vivo, and they suggest that these promoter constructs may be useful for targeted expression of heterologous genes to specific regions of the central and peripheral nervous systems in an inducible manner.