Integrated analysis of genomic and epigenomic instability for CHO cell line engineering

Stability is an important factor in the development of cell lines for therapeutic protein production. In culture, the chromosome number and structure of Chinese Hamster Ovary (CHO) cells undergo rapid change. Over the course of cultivation, selection, and adaptation, chromosomal aberrations such as mutations, deletions, duplications, and other structural variants can accumulate. Some genomic regions may be more prone to such instability than others. When introducing exogenous genes for product formation or for engineering cell characteristics, it is critical to integrate into a stable region. A deeper understanding of the relationship between structure and stability is important for cell culture engineering. We investigated the genome stability of CHO cell lines at the macroscopic and microscopic levels, as well as from the epigenetic and genetic perspective. At the macroscopic level, we examined chromosomal and karyotypic variation, observing that the progenies of single cell clones quickly developed widely distributed variants with different numbers and types of chromosomes. However, at the population level the karyotype and chromosomal number distribution remained in a similar range. Stability at the microscopic level was analyzed using a gene-coding region focused comparative genomic hybridization (CGH) microarray, allowing us to determine genomic variations in gene copy number. With CGH data for many parent-daughter relationships, including subclones and relationships between host and producing cell lines, we identified genome segment changes that happen commonly during cell line development and subcloning. To further examine variation at the microscopic and genetic level, whole genome sequencing data of multiple CHO cell lines was used to identify structural variants, such as deletions, inversions, and duplications using the tools DELLY2 and LUMPY. Heterogeneity was present within each cell line and visible in the form of genome mosaicism. The effect of epigenetic modifications on the CHO genome was explored using the Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq), which examines chromatin accessibility. ATAC-seq information was incorporated with transcriptional activity data using RNA-seq from multiple cell lines to identify inaccessible regions of the genome. This integrated systems approach combining chromosome number, karyotyping, CGH, genome sequencing, ATAC-seq, and RNA-seq gives us insight into the heterogeneity and instability of CHO cells, allowing us to identify desirable and undesirable regions for gene integration. With this data, we can select sites ideal for targeted integration of transgenes as well as screen out potentially unstable cell lines developed using random integration

Sensitivity Analysis of Sidelobes of the Lowest Order Cladding Mode of Long Period Fiber Gratings at Turn Around Point

A new methodology to enhance the sensitivity of a long period fiber grating sensor (LPFG) at the Turn Around Point (TAP) is here presented. The LPFG sensor has been fabricated by etching the fiber up to 20.4 mu m, until the sidelobes of dispersed LP0,2 cladding mode appeared near TAP in aqueous medium. The dual peak sensitivity of the sidelobes was found to be 16,044 nm/SRIU (surrounding refractive index units) in the RI range from 1.333 to 1.3335

対流圏に見られる鉛直微細構造

We report the design, synthesis, detailed characterization, and analysis of a new multifunctional pi-conjugated bola-amphiphilic chromophore: oligo-(p-phenyleneethynylene)-dicarboxylic acid with dialkoxyoctadecyl side chains (OPE-C-18-1). OPE-C-18-1 shows two polymorphs at 123 K (OPE-C-18-1') and 373 K (OPE-C-18-1 `'), whose crystal structures were characterized via single crystal X-ray diffraction. OPE-C-18-1 also exhibits thermotropic liquid crystalline property revealing a columnar phase. The inherent pi-conjugation of OPE-C-18-1 imparts luminescence to the system. Photoluminescence measurements on the mesophase also reveal similar luminescence as in the crystalline state. Additionally, OPE-C-18-1 shows mechano-hypsochromic luminescence behavior. Density functional theory (DFT)-based calculations unravel the origins behind the simultaneous existence of all these properties. Nanoindentation experiments on the single crystal reveal its mechanical strength and accurately correlate the molecular arrangement with the liquid crystalline and mechanochromic luminescence behavior

A synthetic biology based cell line engineering pipeline

An ideal host cell line for deriving cell lines of high recombinant protein production should be stable, predictable, and amenable to rapid cell engineering or other forms of phenotypical manipulation. In the past few years we have employed genomic information to identify “safe harbors” for exogenous gene integration in CHO cells, deployed systems modeling and optimization to design pathways and control strategies to modify important aspects of recombinant protein productivity, and established a synthetic biology approach to implement genetic changes, all with the goal of creating a pipeline to produce “designer” cell lines. Chinese hamster ovary (CHO) cells are the preferred platform for protein production. However, the Chinese hamster genome is unstable in its ploidy, is subject to long and short deletions, duplications, and translocations. In addition, gene expression is subject to epigenetic changes including DNA methylation, histone modification and heterochromatin invasion, thus further complicating transgene expression for protein production in cell lines. With these issues in mind, we set out to engineer a CHO cell line highly amenable to stable protein production using a synthetic biology approach. We compiled karyotyping and chromosome number data of several CHO cell lines and sublines, identified genomic regions with high a frequency of gain and loss of copy number using comparative genome hybridization (CGH), and verified structural variants using sequencing data. We further used ATAC (Assay for Transposase-Accessible Chromatin) sequencing to study chromatin accessibility and epigenetic stability within the CHO genome. RNA-seq data from multiple cell lines were also used to identify regions with high transcriptional activity. Analysis of these data allowed the identification of several “safe harbor” loci that could be used for cell engineering. Based on results of the data analysis and identification of “safe harbors”, we engineered an IgG producing cell line with a single copy of the product transgene as a template cell line. This product gene site is flanked by sequences for recombinase mediated cassette exchange, therefore allowing easy substitution of the IgG producing gene for an alternative product gene. Furthermore, a “landing pad” for multi-gene cassette insertion was integrated into the genome at an additional site. Together, these sites allowed engineering of new cell lines producing a fusion protein and Erythropoietin to be generated from the template cell line. To enable rapid assembly of product transgenes and genetic elements for engineering cell attributes into multi-gene cassettes, we adopted a golden-gate based synthetic biology approach. The assembly of genetic parts into multi-gene cassettes in a LEGO-like fashion allowed different combinations of genes under the control of various promoters to be generated quickly for introduction into the template cell line. Using this engineered CHO cell line, we set out to study metabolism and product protein glycosylation for cell engineering. To guide the selection of genetic elements for cell engineering, we developed a multi-compartment kinetic model, as well as a flux model of energy metabolism and glycosylation. The transcriptome meta-data was used extensively to identify genes and isoforms expressed in the cell line and to estimate the enzyme levels in the model. The flux model was used to identify and the LEGO-like platform was used to implement the genetic changes that can alter the glycosylation pattern of the IgG produced by the template cell line. Concurrently we employed a systems optimization approach to identify the genetic alterations in the metabolic pathway to guide cell metabolism toward a favorable state. The model prediction is being implemented experimentally using the synthetic biology approach. In conclusion, we have illustrated a pipeline of rational cell line engineering that integrates genomic science, systems engineering and synthetic biology approaches. The promise, the technical challenges and possible limitations will be discussed in this presentation

Investigations on the add-layer sensitivity near mode transition of a stretched mode long period fiber grating

The influence of the spectral separation between two consecutive resonant cladding modes of long period fiber grating (LPFG) on the add-layer sensitivity around mode transition (MT) has been investigated with a view to enumerating the dependency of the sensitivity on the separation between the resonant wavelengths of cladding modes. The separation between two consecutive resonant modes was increased or stretched by etching, and the specific mode of interest was brought to the MT region by the deposition of electrostatic self-assembled (ESA) layers of polymeric material on the sensor surface. The add-layer sensitivity of the mode-stretched LPFG (MSLPFG) operating around the linear part of the MT region was found to be similar to 2.59 nm(WL)/nm(TH). The result was found to be superior almost by 1.7 times as compared to a similar LPFG sensor at MT, but without mode stretching. Theoretical modelling has also been shown to corroborate the experimental results