ABSTRACT
As a host for therapeutic protein expression, Chinese hamster ovary (CHO) cells are widely utilized in the mainstream biopharmaceutical industry. Cell culture process development plays an important role in transitioning laboratory research to manufacturing. Among different mathematic tools, kinetic modeling is commonly achieved through analyzing cell culture data to design process parameters, optimize media, and scale up bioreactors. In this review, we examine key factors for upstream process development, and summarize currently used kinetic modeling strategies. In addition, two original examples of kinetic modeling application optimizing cell culture performance are presented. A comprehensive understanding is provided for the kinetic modeling and its applications in cell culture process development.
Subject(s)
Bioreactors , Proteomics , Animals , CHO Cells , Cell Culture Techniques , Cricetinae , Cricetulus , KineticsABSTRACT
Alternative cleavage and polyadenylation (APA) can occur at more than half of all human genes, greatly enhancing the cellular repertoire of mRNA isoforms. As these isoforms can have altered stability, localisation and coding potential, deregulation of APA can disrupt gene expression and this has been linked to many diseases including cancer progression. How APA generates cancer-specific isoform profiles and what their physiological consequences are, however, is largely unclear. Here we use a subcellular fractionation approach to determine the nuclear and cytoplasmic APA profiles of successive stages of colon cancer using a cell line-based model. Using this approach, we show that during cancer progression specific APA profiles are established. We identify that overexpression of hnRNPC has a critical role in the establishment of APA profiles characteristic for metastatic colon cancer cells, by regulating poly(A) site selection in a subset of genes that have been implicated in cancer progression including MTHFD1L.
Subject(s)
Alternative Splicing , Heterogeneous-Nuclear Ribonucleoprotein Group C/genetics , Neoplasms/genetics , Polyadenylation , RNA Isoforms/genetics , Aminohydrolases/genetics , Aminohydrolases/metabolism , Cell Line, Transformed , Cell Line, Tumor , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Disease Progression , Formate-Tetrahydrofolate Ligase/genetics , Formate-Tetrahydrofolate Ligase/metabolism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Heterogeneous-Nuclear Ribonucleoprotein Group C/metabolism , Humans , Methylenetetrahydrofolate Dehydrogenase (NADP)/genetics , Methylenetetrahydrofolate Dehydrogenase (NADP)/metabolism , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Poly A/genetics , Poly A/metabolism , RNA InterferenceABSTRACT
Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3'end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease.
