Cellule CHO

vignette|Cellules CHO vues au microscope à contraste de phase. Les cellules CHO sont une lignée cellulaire issue d'ovaires de hamster de Chine (Cricetulus griseus) couramment utilisée en recherche dans le domaine médical, en biotechnologie et par l'industrie pharmaceutique pour la production de protéines thérapeutiques. Elles ont été introduites dans les années 1960, à partir d'une lignée isolée en 1957, comme culture cellulaire monocouche nécessitant l'adjonction de proline dans leur milieu de culture. Elles existent aujourd'hui selon différentes lignées présentant des altérations génétiques variables. Elles sont utilisées pour les études génétiques, les recherches sur l'expression des gènes — notamment pour produire des protéines recombinantes — et la nutrition, ainsi que pour réaliser des analyses de toxicité. Il s'agit de l'une des lignées cellulaires le plus couramment utilisées dans la production de médicaments tels que les anticorps thérapeutiques. Près de 70 % de toutes les

Establishment of recombinant mammalian cell lines

Madiha Derouazi

Recombinant proteins are gaining in importance for therapeutic applications. The proteins are expressed in stable cell lines, within which recombinant DNA has incorporated into the host cells genome. Identification and isolation of extremely high producers from transfected cell populations is a tedious and time-consuming effort. We proposed two different approaches for the establishment of recombinant cell lines: affinity cell sorting and microinjection. Even with the help of single cell sorting and analysis systems (flow cytometry) rarely more than thousand different cell clones, stably expressing a desired gene can be evaluated. We are considering a new approach in which polyclonal cell populations will be established which co-express a protein of interest and an epitope-tagged cell surface receptor. A microbead fixed antibody specific to the epitope tag is hoped to assist in the isolation of rare, but highly productive cells from large suspensions of transfected cells. For this reason we have also developed an efficient method for the bulk transfection of CHO cells. On the side of vector-development for this approach, we have chosen to investigate the utility of a mouse serotonin 5-HT3 receptor as a cell surface marker. This receptor can be expressed at very high levels (up to 1 x 107 receptors per cell) in heterologous systems. The receptor is detectable on the cell surface by means of fluorescent ligands, which allows the determination of transfection efficiencies in suspension cultures. A Flag-tag was fused to the amino terminal end of the receptor without adverse effects on receptor function or ligand binding. Only 5 % of receptor DNA in the transfection cocktail was sufficient to detect positive cells in a population. The specific interaction of the epitope-tagged receptor with the cognate antibody fixed to microbeads was tested in comparison to non-transfected control cells using laser scanning confocal microscopy. A 80% enrichment of cells expressing GFP and the 5-HT3 receptor was achieved in 30 minutes. We have investigated microinjection as a gene transfer method for the establishment of recombinant mammalian cell lines. The conditions for the injection of plasmid DNA into either the nucleus or the cytoplasm of CHO-DG44 cells were established using pMYK-EGFP, an expression vector with the enhanced green fluorescent protein (EGFP) gene under the control of the murine CMV promoter and the puromycin resistance gene for selection. To estimate plasmid copy number during microinjection, it was first necessary to determine the injection volume by fluorescence quantification of injected FITC-dextran. For microinjection into the cytoplasm the volume ranged from 200 to 350 fl and for nuclear microinjection it was about 180 fl. We then investigated the impact of DNA concentration and injection pressure on transient EGFP transient expression following microinjection into the cytoplasm or nucleus. As a general rule, we observed that concentration of the DNA solution injected into the cells was the most important parameter regardless of the pressure or the volume of injection. Recombinant cell lines expressing intracellular EGFP were established following microinjection into the nucleus and the cytoplasm with linear and circular DNA. Selection with puromycin, clonal expansion, and cell banking were completed within three to four weeks post-microinjection. In parallel, recombinant cell lines were established after calcium-phosphate transfection with linear and circular DNA. The number of plasmid copies integrated was determined by Southern blot and the number and localization of integration sites was determined by fluorescence in-situ hybridization (FISH). Growth of the cells and stability of EGFP expression were investigated by cultivation over a two-month period. This approach presents several advantages over other DNA delivery methods such as control of the quantity and localization of DNA delivered into the cell and speed of recovery of recombinant cell lines. This approach may be relevant for the establishment of recombinant cell lines from cells that are not easily transfected by classical methods.

EPFL2005

Gamma-Secretase Dependent Gene Expression

Alexandra Marie-Isabelle Karoline Brigitte Magold

The causes of sporadic Alzheimer's Disease (AD), the most common form of dementia at old age, are still unknown. Familial early onset AD (FAD) is mainly caused by mutations of Presenilins (PS), the active subunits of γ-secretase. Processing of its substrate proteins by γ-secretase generates shorter fragments. In the case of the Amyloid Precursor Protein (APP) these are Aβ species, of which the ratio of Aβ40, which is 40 amino acids (aa) long and Aβ42, 42 aa in length respectively, has been connected with their aggregation and AD plaque formation. Another cleavage product of many γ-secretase substrate proteins are intracellular domains (ICDs), which enter the nucleus and alter gene transcription. Be it via statins, the levels of cAMP, the generation of which is also catalyzed by cilia specific Adenylyl-cyclase III (ACIII) or Wnt pathways, cilia can be connected with various AD relevant proteins, pathways and functions, but their physiological and thus pathological cerebral roles are not understood. We hypothesized that γ-secretase activity alterations impact transcription levels of specific genes and potentially signaling and functional pathways as well as cilia genes in ways relevant to AD. We determined the transcriptional impact of enhanced γ-secretase activity in Chinese hamster ovary (CHO) cells with a cDNA microarray experiment. Gene ontology cluster analysis of the 1981 genes reported by our microarray gave insights into functional groups represented. Matching of known physical protein-protein interactions as reported by the string 8.0 database for 21 genes, which were confirmed to be transcriptionally altered with γ-secretase activity, registered various members of three different Wnt pathways. Consistent with an hypothesized role of cilia, the array also reported two dozen of cilia-related genes. The most decreased transcript levels were of Anti-mitotic exit network homolog 1 (AMN1), a gene of predicted expression in the cilia of sensory olfactory neurons and for which protein data was available in yeast only. Our immunohistochemistry results confirm Amn1 protein expression in mammalian cells, specifically in murine and human brains. We showed that Amn1 protein is localized in cortical neurons and in sharply outlined intracellular structures close to motile cilia and the opposing cell pole of monocilia. Aged APPPS1 transgenic mice, which over express human APP and PS1 with FAD mutations, did neither show differences in lateral ventricular cilia range, cilia density, the percentage of ciliated hippocampal neurons nor ACIII protein levels. Consistently with previous reports of increased cAMP levels in the cerebrospinal fluid of AD patients, we detected significantly increased levels of ACIII protein in human AD cortex samples (AD n=11, non demented n=12). These results support a new focus on the role of cilia-related proteins in AD. They also suggest some limitations of the APPPS1 transgenic animal model. In recent years the impact and role of cilia-related proteins in various neurodegenerative diseases was demonstrated. This PhD thesis provides evidence that AD should be included in the list of neurodegenerative diseases which are being investigated for the impact and role of cilia-related proteins in their pathologies.

EPFL2011

Gamma-Secretase Dependent Gene Expression

Alexandra Marie-Isabelle Karoline Brigitte Magold

EPFL2011

Establishment of recombinant mammalian cell lines

Madiha Derouazi

EPFL2005

Characterization of axial-flow packed bed bioreactors for the culture of CHO cells

Establishment of recombinant mammalian cell lines

Gamma-Secretase Dependent Gene Expression

Characterization of axial-flow packed bed bioreactors for the culture of CHO cells

Gamma-Secretase Dependent Gene Expression

Establishment of recombinant mammalian cell lines