Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next-Generation Sequencing Technologies for Cell Line Development.

Early analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next-generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single-nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high-level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays.

The centriolar satellite protein SSX2IP promotes centrosome maturation.

Meiotic maturation in vertebrate oocytes is an excellent model system for microtubule reorganization during M-phase spindle assembly. Here, we surveyed changes in the pattern of microtubule-interacting proteins upon Xenopus laevis oocyte maturation by quantitative proteomics. We identified the synovial sarcoma X breakpoint protein (SSX2IP) as a novel spindle protein. Using X. laevis egg extracts, we show that SSX2IP accumulated at spindle poles in a Dynein-dependent manner and interacted with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1. Immunodepletion of SSX2IP impeded γ-TuRC loading onto centrosomes. This led to reduced microtubule nucleation and spindle assembly failure. In rapidly dividing blastomeres of medaka (Oryzias latipes) and in somatic cells, SSX2IP knockdown caused fragmentation of pericentriolar material and chromosome segregation errors. We characterize SSX2IP as a novel centrosome maturation and maintenance factor that is expressed at the onset of vertebrate development. It preserves centrosome integrity and faithful mitosis during the rapid cleavage division of blastomeres and in somatic cells.

Centriolar satellites: busy orbits around the centrosome.

Since its first description by Theodor Boveri in 1888, the centrosome has been studied intensely, and it revealed detailed information about its structure, molecular composition and its various functions. The centrosome consists of two centrioles, which generally appear in electron microscopy as barrel-shaped structures usually composed of nine microtubule triplets. An amorphous mass of pericentriolar material surrounds the centrioles and accumulates many proteins important for the integrity and function of centrosomes, such as the γ-tubulin ring complex (γ-TuRC) that mediates microtubule nucleation and capping. In animal somatic cells, the centrosome generally accounts for the major microtubule organizing center, and the duplicated pair of centrosomes determines the poles of the microtubule-based mitotic spindle. Despite detailed insights into the centrosome's structure and function, it has been a complete mystery until a few years ago how centrosomes duplicate and assemble. Moreover, it is still largely unclear if and how centrosomal proteins or protein complexes are exchanged, replaced or qualitatively altered. Previously identified cytoplasmic granules, named "pericentriolar" or "centriolar satellites", might fulfil such functions in protein targeting and exchange, and communication between the centrosomes and the cytoplasm. In this review, we summarize current knowledge about the structure, molecular composition and possible roles of the satellites that seem to surround the core of the centrosome in most animal cells.

Monoclonal antibodies with selective specificity towards different glycosylation isoforms of FGFR1.

Fibroblast growth factor receptor 1 (FGFR1) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes including cell proliferation, differentiation, survival, and tumorigenesis. This diversity is possibly mediated by the existence of multiple FGFR1 isoforms, generated by alternative splicing and post-translational modifications, mainly through glycosylation. In this study we report the generation and characterization of a panel of monoclonal antibodies directed towards FGFR1. To achieve this, we used as an antigen a fragment of FGFR1, corresponding to loop II-III of the extracellular domain, which shares low homology to other members of the FGFR family and possesses numerous antigentic determinants. Two rounds of ELISA screening and Western blot analysis allowed us to isolate a panel of monoclonal antibodies, which recognize specifically recombinant FGFR1 loop II-III. The ability of generated antibodies to recognize endogenous FGFR1 was examined in 3T3 L1 cells, which are known to express FGFR1, but not other members of FGFR family. Immunoblot analysis of 3T3 L1 cell lysates with hybridoma media of selected clones revealed a different, but overlapping pattern of immunoreactive bands, which might represent splicing and post-translationally modified forms of FGFR1. Furthermore, we also tested the cross-reactivity of generated antibodies towards recombinant full-length FGFR3 and their ability to recognize FGFR1 in 3T3 L1 cells by cyto- and immunocytochemistry. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR1 in normal and pathological tissues.