Single-Cell Analysis Reveals that the Enterococcal Sex Pheromone Response Results in Expression of Full-Length Conjugation Operon Transcripts in All Induced Cells.

For high-frequency transfer of pCF10 between E. faecalis cells, induced expression of the pCF10 genes encoding conjugative machinery from the prgQ operon is required. This process is initiated by the cCF10 (C) inducer peptide produced by potential recipient cells. The expression timing of prgB, an "early" gene just downstream of the inducible promoter, has been studied extensively in single cells. However, several previous studies suggest that only 1 to 10% of donors induced for early prgQ gene expression actually transfer plasmids to recipients, even at a very high recipient population density. One possible explanation for this is that only a minority of pheromone-induced donors actually transcribe the entire prgQ operon. Such cells would not be able to functionally conjugate but might play another role in the group behavior of donors. Here, we sought to (i) simultaneously assess the presence of RNAs produced from the proximal (early induced transcripts [early Q]) and distal (late Q) portions of the prgQ operon in individual cells, (ii) investigate the prevalence of heterogeneity in induced transcript length, and (iii) evaluate the temporality of induced transcript expression. Using fluorescent in situ hybridization chain reaction (HCR) transcript labeling and single-cell microscopic analysis, we observed that most cells expressing early transcripts (QL, prgB, and prgA) also expressed late transcripts (prgJ, pcfC, and pcfG). These data support the conclusion that, after induction is initiated, transcription likely extends through the end of the conjugation machinery operon for most, if not all, induced cells.IMPORTANCE In Enterococcus faecalis, conjugative plasmids like pCF10 often carry antibiotic resistance genes. With antibiotic treatment, bacteria benefit from plasmid carriage; however, without antibiotic treatment, plasmid gene expression may have a fitness cost. Transfer of pCF10 is mediated by cell-to-cell signaling, which activates the expression of conjugation genes and leads to efficient plasmid transfer. Yet, not all donor cells in induced populations transfer the plasmid. We examined whether induced cells might not be able to functionally conjugate due to premature induced transcript termination. Single-cell analysis showed that most induced cells do, in fact, express all of the genes required for conjugation, suggesting that premature transcription termination within the prgQ operon does not account for failure of induced donor cell gene transfer.

Multiplexed clonality verification of cell lines for protein biologic production.

During the development of cell lines for therapeutic protein production, a vector harboring a product transgene is integrated into the genome. To ensure production stability and consistent product quality, single-cell cloning is then performed. Since cells derived from the same parental clone have the same transgene integration locus, the identity of the integration site can also be used to verify the clonality of a production cell line. In this study, we present a high-throughput pipeline for clonality verification through integration site analysis. Sequence capture of genomic fragments that contain both vector and host cell genome sequences was used followed by next-generation sequencing to sequence the relevant vector-genome junctions. A Python algorithm was then developed for integration site identification and validated using a cell line with known integration sites. Using this system, we identified the integration sites of the host vector for 31 clonal cell lines from five independent vector integration events while using one set of probes against common features of the host vector for transgene integration. Cell lines from the same lineage had common integration sites, and they were distinct from unrelated cell lines. The integration sites obtained for each clone as part of the analysis may also be used for clone selection, as the sites can have a profound effect on the transgene's transcript level and the stability of the resulting cell line. This method thus provides a rapid system for integration site identification and clonality verification.

Cell culture bioprocessing - the road taken and the path forward.

Cell culture processes are used to produce the vast majority of protein therapeutics, valued at over US$180 billion per annum worldwide. For more than a decade now, these processes have become highly productive. To further enhance capital efficiency, there has been an increase in the adoption of disposable apparatus and continuous processing, as well as a greater exploration of in-line sensing, various -omic tools, and cell engineering to enhance process controllability and product quality consistency. These feats in cell culture processing for protein biologics will help accelerate the bioprocess advancements for virus and cell therapy applications.

Recurring genomic structural variation leads to clonal instability and loss of productivity.

Chinese hamster ovary cells, commonly used in the production of therapeutic proteins, are aneuploid. Their chromosomes bear structural abnormality and undergo changes in structure and number during cell proliferation. Some production cell lines are unstable and lose their productivity over time in the manufacturing process and during the product's life cycle. To better understand the link between genomic structural changes and productivity stability, an immunoglobulin G producing cell line was successively single-cell cloned to obtain subclones that retained or lost productivity, and their genomic features were compared. Although each subclone started with a single karyotype, the progeny quickly diversified to a population with a distribution of chromosome numbers that is not distinctive from the parent and among subclones. The comparative genomic hybridization (CGH) analysis showed that the extent of copy variation of gene coding regions among different subclones stayed at levels of a few percent. Genome regions that were prone to loss of copies, including one with a product transgene integration site, were identified in CGH. The loss of the transgene copy was accompanied by loss of transgene transcript level. Sequence analysis of the host cell and parental producing cell showed prominent structural variations within the regions prone to loss of copies. Taken together, we demonstrated the transient nature of clonal homogeneity in cell line development and the retention of a population distribution of chromosome numbers; we further demonstrated that structural variation in the transgene integration region caused cell line instability. Future cell line development may target the transgene into structurally stable regions.

Single Copy Transgene Integration in a Transcriptionally Active Site for Recombinant Protein Synthesis.

For the biomanufacturing of protein biologics, establishing stable cell lines with high transgene transcription is critical for high productivity. Modern genome engineering tools can direct transgene insertion to a specified genomic locus and can potentially become a valuable tool for cell line generation. In this study, the authors survey transgene integration sites and their transcriptional activity to identify characteristics of desirable regions. A lentivirus containing destabilized Green Fluorescent Protein (dGFP) is used to infect Chinese hamster ovary cells at a low multiplicity of infection, and cells with high or low GFP fluorescence are isolated. RNA sequencing and Assay for Transposase Accessible Chromatin using sequencing data shows integration sites with high GFP expression are in larger regions of high transcriptional activity and accessibility, but not necessarily within highly transcribed genes. This method is used to obtain high Immunoglobulin G (IgG) expressing cell lines with a single copy of the transgene integrated into transcriptionally active and accessible genomic regions. Dual recombinase-mediated cassette exchange is then employed to swap the IgG transgene for erythropoietin or tumor necrosis factor receptor-Fc. This work thus highlights a strategy to identify desirable sites for transgene integration and to streamline the development of new product producing cell lines.

Stochasticity in the enterococcal sex pheromone response revealed by quantitative analysis of transcription in single cells.

In Enterococcus faecalis, sex pheromone-mediated transfer of antibiotic resistance plasmids can occur under unfavorable conditions, for example, when inducing pheromone concentrations are low and inhibiting pheromone concentrations are high. To better understand this paradox, we adapted fluorescence in situ hybridization chain reaction (HCR) methodology for simultaneous quantification of multiple E. faecalis transcripts at the single cell level. We present direct evidence for variability in the minimum period, maximum response level, and duration of response of individual cells to a specific inducing condition. Tracking of induction patterns of single cells temporally using a fluorescent reporter supported HCR findings. It also revealed subpopulations of rapid responders, even under low inducing pheromone concentrations where the overall response of the entire population was slow. The strong, rapid induction of small numbers of cells in cultures exposed to low pheromone concentrations is in agreement with predictions of a stochastic model of the enterococcal pheromone response. The previously documented complex regulatory circuitry controlling the pheromone response likely contributes to stochastic variation in this system. In addition to increasing our basic understanding of the biology of a horizontal gene transfer system regulated by cell-cell signaling, demonstration of the stochastic nature of the pheromone response also impacts any future efforts to develop therapeutic agents targeting the system. Quantitative single cell analysis using HCR also has great potential to elucidate important bacterial regulatory mechanisms not previously amenable to study at the single cell level, and to accelerate the pace of functional genomic studies.