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A platform for Chinese hamster ovary (CHO) cell genome engineeringDoshi, Jiten January 2016 (has links)
The production of therapeutic recombinant proteins in heterologous systems has gained significance since the last decade. For recombinant proteins that require post-translational modifications (PTMs), mammalian systems are preferred. Chinese hamster ovary (CHO) cells are the mammalian cells of choice for production of recombinant proteins. This is because of their ability to provide correct protein-folding and post-translational modifications, displaying high productivity at large scale, ability to grow in suspension mode at high densities in a serum-free media, incapable of infection by most viruses and their history of regulatory approvals. There is an established state of the art technology for development of CHO cells for recombinant protein production. This technology relies on random integration of the gene of interest and gene amplification process for obtaining high expressing clones. There is a high degree of clonal heterogeneity and instability observed in the screened clones. To overcome the process of random integration, this report describes a lentivirus based screening for search of stable and high expressing integration sites in CHO cells. The integration sites are identified by using nrLAM-PCR (non-restrictive linear amplification mediated PCR) coupled with high throughput sequencing. Lentivirus are chosen as they preferentially integrate within the coding regions rendering the possibility of obtaining stable and high expressing clones. In addition, lentivirus vector is designed to possess landing pad for recombinase mediated cassette exchange of viral sequence with foreign DNA. The report describes a successful cassette exchange reaction but with low efficiency. Genome engineering technologies such as CRISPR/Cas, TALENs can used for targeted gene insertion at integration sites and thus establishing stable and efficient production of recombinant proteins in CHO cells. Additionally, an approach for designing synthetic promoters based on Ef1α promoter architecture has been shown. Synthetic promoters are useful for expression of multi-gene cassettes as they are short in length and provide comparable expression levels to the native mammalian promoter.
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Investigating the influence of long-term culture and feed additions on recombinant antibody production in Chinese hamster ovary cellsBailey, Laura January 2011 (has links)
Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies (MAbs), due to their ability to perform correct post-translational modifications. A major issue for use of CHO cells lines for the production of recombinant proteins is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval. Protein production is complex and is influenced by cell growth, transcription, translation, protein folding and post-translational processing and secretory events, which may interact to determine stability of expression during prolonged culture. This thesis aims to identify features associated with stability/instability of recombinant protein expression and methods to improve protein production, with the addition of chemically defined (CD) feed and chemicals. Two exemplar CHO cell lines, which secrete the same recombinant antibody were characterised in response to LTC, feed and DMSO addition. Both cell lines (3.90 and 51.69) exhibited unstable protein production over LTC, with a loss in final antibody titres and specific productivity (Qp). The instability observed within the exemplar cell lines was not due to decreased recombinant gene copy numbers or mRNA expression but was associated with lower viable cell densities, increased ER stress (GADD153 and spliced XBP-1 [XBP-1(s)]) and enhanced rates of lactate utilisation (observed during the decline phase of batch culture). Improvement of recombinant protein expression in response to feed or DMSO addition was associated with lower expression of ER stress markers (ATF4, XBP-1(s) and GADD153 at mRNA level and GADD153 at protein level) and alterations to the metabolic activity of the cultures (prevention of alanine and lactate re-utilisation, and greater glucose utilisation between the stationary and decline phase of batch culture).Although feed or DMSO addition improved recombinant protein production, these additions did not reverse the appearance or progression of instability for cells after LTC. ER stress expression was not abolished as a consequence of feed or DMSO addition. Expression of stress markers at earlier time points may be the factor that limits antibody production and secretion. The consequences of the presence of feed and DMSO addition on ER stress markers and antibody production serves to highlight approaches that may be utilised for engineering more productive or stable protein production phenotypes in parental cell lines.
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Proteomic Analysis of Chinese Hamster Ovary Cells Producing Glycosylated Monoclonal AntibodiesHo, Raymond January 2013 (has links)
Therapeutic monoclonal antibodies (MAb) are produced as secreted complex glycoproteins from mammalian cell systems and represent one of the most important classes of therapeutic medicines for the treatment of a variety of human diseases. Their benefit in health care and high economic impact provide the driving force for the development of improved production levels with the focus of optimizing clinical efficacy. One important issue is the optimization of monoclonal antibody production. A frequent approach used to address this challenge is the engineering of mammalian cell lines to increase antibody production levels through genetic manipulation. Valuable information can then be obtained by monitoring the effects of genetic changes on the biochemistry of the cell associated with MAb production. Global protein expression profiling of mammalian cells used for the production of biopharmaceuticals may reveal key biochemical characteristics associated with MAb-producing cell lines. A better understanding of these characteristics can in turn lead to more rational strategies for cell line and process development.
The proposed research relates to a larger NSERC Strategic Network (MAbNet) Grant to develop and establish a novel platform for the large-scale manufacture of specific glycoforms of therapeutic monoclonal antibodies. The efficacy of these recombinant MAbs will be enhanced by the control of their glycosylation profiles. The work presented in this thesis will assist MAbNet in meeting their objectives. Specifically, we use 2D-Differential In-Gel Electrophoresis (2D-DIGE) to quantify protein expression differences between EG2-hFc1-producing Chinese Hamster Ovary cells (CHO-1A7) with its parental cell line (CHO-BRI). Here, we identified 34 unique differentially expressed proteins associated with EG2-hFc1 production that relate to various biological processes including protein processing, carbohydrate metabolism, amino acid metabolism, energy metabolism, apoptosis, and cell proliferation pathways.
The majority of identified significant protein expression changes and their associated metabolic processes seem to prioritize energy production in CHO-1A7 cells. Due to the metabolic load of recombinant antibody production, the CHO-1A7 cell line attempts to meet the energy requirements needed for recombinant protein biosynthesis while maintaining cell viability and efficient protein folding mechanisms.
A 2-D proteome reference map was also constructed for the CHO-BRI host cell line containing 131 identified protein spots. The map provides information that will further expand our understanding of this particular cell line. It will be a useful tool for studies investigating physiological responses and protein expression patterns of CHO-BRI to genetic and environmental perturbations.
The set of identified differentially expressed proteins provides data on the downstream changes in protein expression due to genetic manipulation, and furthermore can provide targets for cell-line specific optimization of antibody production. The work described in this thesis furthers our understanding of antibody production in a specific CHO cell line.
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Transient production of biopharmaceutical proteinsWei, Tzu-Hsiang, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2009 (has links)
The creation of stable mammalian cell lines for biopharmaceutical production often require several months, and is unfavourable for the rapid production of multiple drug candidates for screening in the early stages of development. Biopharmaceutical production by transient transfection provides a possible alternative of quickly producing these early stage drug candidates. The Epi-CHO transient expression system, which consists of a Chinese hamster ovary (CHO) cell line (CHO-T) expressing the murine polyomavirus Large T-Antigen (LT), emonstrated enhanced transient recombinant protein production. The aim of this study was to prolong transient recombinant protein prod.Jction of the Epi-CHO expression system by creating a CHO cell line expressing both LT and EBNA1 (ECHO-T). The pEBNA1-LT expression vector encoding LT and EBNA1 was constructed and transfected into CHO-K1. A total of 20 clones were isolated from the antibioticresistant pool and screened for the expression of functional LT and EBNA1. PCR analysis showed 16 of the 20 clones was positive for EBNA1 and LT DNA. Of the 16 clones, six were positive for EBNA1 and LT expression by RT-PCR. Detection of LT and EBNA1 by immunofluorescence showed positive staining for the P7-G3 clone. Western blotting suggested the P7-G3 clone was: positive for EBNA1, and clones P3-C7 and P7-E2 were positive for LT. A plasmid replication assay confirmed the expression of functional LT in all six clones. Plasmid maintenance assay confirmed clone P7-G3 as the ECHO-T clones to express functional EBNA1. The P7-G3 clone demonstrated prolonged and sustained transient recombinant protein expression when compared to CHO-T. The P7-G3 clone achieved sustained transient protein expression for 32 days in the absence of selection, the longest currently reported for CHO cells.
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The Role of Nucleotide Excision Repair Genes in the Repair of Methylene Blue Plus Visible Light-Induced DNA and Cellular Damage in Chinese Hamster Ovary CellsCowan, Robert 01 1900 (has links)
<p>Base excision repair (BER) is a DNA repair mechanism that involves the removal of single damaged bases from DNA and their excision as free bases. Another DNA repair mechanism known as nucleotide excision repair (NER) involves the removal of bulky lesions from DNA. Previous published reports have suggested a role for certain NER proteins in BER. Methylene blue (MB) is a type II photosensitizer, which upon excitation by visible light (VL) produces singlet oxygen that reacts with DNA to form 8hydroxyguanine (8-oxoG) lesions. The BER mechanism has been shown to preferentially remove 8-oxoG lesions from DNA. In the current work, the role of NER proteins in the BER of [MB+VL]-induced DNA damage was examined using a reporter gene assay. AdMCMVlacZ and AdHCMVlacZ are non-replicating adenoviruses that express the bacterial β-galactosidase ( β-gal) reporter gene under the control of the murine or human cytomegalovirus immediate early promoter, respectively. Host cell reactivation (HCR) of β-gal activity for a [MB+VL]-treated AdMCMVlacZ was examined in two repair-proficient normal Chinese hamster ovary (CHO) cell lines, NER-deficient mutants from rodent complementation groups (RCGs) 1 through 6, and the HER-deficient cell line EM9. Results show a decreased HCR capacity of [MB+VL]-induced DNA damage in the UV61 (RCG-6; CSB) cell line when compared to the repair-proficient normal AA8. This suggests an involvement of CSB in BER of [MB+VL]-induced damage. In contrast, the XRCCl-deficient EM9 showed an increased HCR capacity when compared to the parental AA8. This suggests a beneficial role for an XRCCl deficiency or for the specific gain-of-function gene mutation in the XRCCl gene for the repair of [MB+VL]induced damage. Similarly, the ERCCl-deficient UV20 (RCG-1) showed an increased HCR capacity when compared to the parental AA8. In contrast, another ERCCJdeficient cell line, 30PV, and the ERCCJ knock-out cell line, CH0-7-27, showed no significant increase in β-gal activity when compared to the parental CHO-Kl. The ability to induce BER of [MB+VL]-induced DNA damage was also examined. HCR of β-gal activity for [MB+VL]-treated AdHCMVlacZ or AdMCMVlacZ was examined in several CHO cell lines that were either untreated or treated with low levels of UVC or MB plus VL. Pre-treatment of NER-deficient UV61 and repair-proficient AA8 cells with UVC resulted in an enhanced HCR for [MB+VL]-treated AdHCMVlacZ, although the results were only significant for UV61. Similarly, an enhanced HCR of [MB+VL]-treated AdMCMVlacZ was observed in AA8 cells, suggesting that the repair of the [MB+VL]treated reporter gene is inducible by UVC. A significant enhancement in HCR of [MB+VL]-treated AdMCMV lacZ was not observed in AA8 cells following pre-treatment with MB, VL, or both, indicating that the detection of any induction in the repair of DNA damage from MB plus VL could not be made with the conditions of the HCR assay employed. As investigations into the effects of MB plus VL on whole cells have been limited, clonogenic survival of BER-and NER-deficient CHO cell lines was also observed following treatment with MB alone or MB plus VL. Results showed that the sensitivities of the NER-deficient CHO cell lines from RCGs 2, 4 and 5 and the BERdeficient EM9 cell line to MB alone were within the range obtained for the two repairproficient CHO normal cell lines, AA8 and K 1. In contrast, the UV20 cell line was more sensitive to MB alone compared to the normal cell lines. Additionally, both UV24 (RCG-3; XPB) and UV61 showed a decreased sensitivity toMB alone when compared to the normal AA8 cell line. The sensitivity of cells to MB plus VL was greater in the UV24, UV135 (RCG-5; XPG) and the XRCCl-deficient EM9 cell lines compared to that of the range obtained for the two repair-proficient normal cell lines, AA8 and Kl. Taken together with the results from the HCR assays, these results suggest that [MB+VL]induced DNA damage to cells and its repair do not play a major role in the survival of cells following treatment with MB plus VL. It appears likely that damage to cellular components other than DNA, such as protein, lipid and biological membranes, play a more important role in cell killing by MB plus VL.</p> / Thesis / Master of Science (MSc)
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Phosphatidylethanolamine deficiency in mammalian cellsBai, Helin Daniel Unknown Date
No description available.
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Phosphatidylethanolamine deficiency in mammalian cellsBai, Helin Daniel 11 1900 (has links)
Almost all mammalian cells contain energy-producing organelles called mitochondria. Phosphatidylethanolamine (PE) is a phospholipid which has been implicated to be important for mitochondrial function. The majority of mitochondrial PE is synthesized in mitochondria using the phosphatidylserine decarboxylase (PSD) pathway. To test the hypothesis that PE made from the PSD pathway is required for mitochondrial function, three Chinese Hamster Ovary Cell lines with different PSD-pathway defects were studied. These three cell lines referred to as PSB-2, R-41, and PSD knockdown cells all had ~35% reductions in mitochondrial PE levels compared to the parental cell line. As a result, the mitochondria from all three cell lines have abnormally high sedimentation densities and increased membrane potentials. However, the energy production, motility, and morphologies of each type of mutant mitochondria were each distinctly different from their parental cell line. / Experimental Medicine
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Molecular Cloning of a Chinese Hamster Mitochondrial Protein Related to the Chaperonin Family of Proteins / Molecular Cloning of a Chinese Hamster Mitochondrial ProteinPicketts, David 12 1900 (has links)
The complete cDNA sequence of a mitochondrial protein from Chinese hamster ovary cells has been determined. This protein, designated P1, was originally identified in cells resistant to the microtubule inhibitor podophyllotoxin (Gupta, 1981). The mutant cell line contained an alteration of the P1 protein that gave rise to a new, more acidic protein, designated M1 (Gupta et al., 1982). The P1 protein was determined to be microtubule-related based on the cross-resistance pattern of the mutants to other microtubule inhibitors, and corelease with tubulin under conditions which cause microtubule depolymerization (Gupta et al., 1982). Subcellular fractionation studies localized this protein to the matrix of the mitochondria (Gupta and Austin, 1987). Antibodies raised against P1 were used to isolate a cDNA clone from human cells (Jindal et al.,1989). The human cDNA clone was used as a probe to screen for clones of the P1 protein in bacteriophage (lambda)gt10/(lambda)gt11 cDNA libraries prepared from CHO cells. The P1 cDNA encodes a protein of 573 amino acids with a relative molecular mass of 60,983 daltons. The first 26 amino acids meet the requirements of a mitochondrial matrix targeting sequence. The mature protein is 547 residues in length with a relative molecular mass of 57,949 daltons. The deduced amino acid sequence shows 97% identity to the the human P1 protein. More interestingly, the amino acid sequence shows extensive homology (42 to 55% identical residues and an additional 15 to 20% conservative replacements) to the chaperonin class of molecular chaperones. This class of proteins includes the hsp60 protein of yeast, the groEL protein of Escherichia coli, the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit binding protein of plant chloroplasts, and the 62-65-kDa major antigenic protein of mycobacteria and Coxiella burnetii. The homology of P1 with the above proteins begins after the putative mitochondrial presequence and extends to the c-terminal end. Several regions throughout the protein sequence are highly conserved and are proposed to be functional domains of the protein. Also highly conserved is a Gly-Gly-Met repeating motif at the carboxy-terminus. The function of this sequence is undetermined, as yet. A dendrogram was constructed from the sequence homology data. It suggested that mitochondrial P1 evolved from purple bacteria which is the endosymbiont which gave rise to mitochondria. The chaperonin class of proteins have been shown to assist in the assembly of oligomeric protein structures. It is suggested that the P1 protein may play a similar role in mammalian cells. The high degree of homology between P1 and the 65-kDa mycobacterial antigen also suggests that P1 may be involved in certain autoimmune diseases. / Thesis / Master of Science (MS)
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Identification of cellular changes associated with increased production of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell lineMisztal, David Richard, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2008 (has links)
A proteomics approach was used to identify proteins potentially implicated in the cellular response concurrent with elevated production levels of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line (Darren cells), using zinc and sodium butyrate in the production media to increase expression. To this end, 2-dimensional gel electrophoresis (2-DGE) was utilized. Firstly, several aspects of 2-DGE were developed for this investigation. Gel drying conditions were optimized, and a glycine-free blotting method is described which achieved greater efficiency in rapid transfer of proteins than those previously described. Next, hFSH expression was characterized in Darren cells. An ELISA developed for this investigation examined intracellular (expression) and extracellular (secretion) of hFSH during increased expression. These results show a disproportionate increase in intracellular hFSH (188%) expression above extracellular hFSH (41%).
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Over Expression of the CMP-sialic Acid Transporter in Chinese Hamster Ovary Cells Leads to Increased SialylationWong, Niki S.C., Yap, Miranda G.S., Wang, Daniel I.C. 01 1900 (has links)
Most glyco-engineering approaches used to improve quality of recombinant glycoproteins involve the manipulation of glycosyltransferase and/or glycosidase expression. We investigated whether the over expression of nucleotide sugar transporters, particularly the CMP-sialic acid transporter (CMP-SAT), would be a means to improve the sialylation process in CHO cells. We hypothesized that increasing the expression of the CMP-SAT in the cells would increase the transport of the CMP-sialic acid in the Golgi lumen, hence increasing the intra-lumenal CMP-sialic acid pool, and resulting in a possible increase in sialylation extent of proteins being produced. We report the construction of a CMP-SAT expression vector which was used for transfection into CHO-IFNγ, a CHO cell line producing human IFNγ. This resulted in approximately 2 to 5 times increase in total CMP-SAT expression in some of the positive clones as compared to untransfected CHO-IFNγ, as determined using real-time PCR analysis. This in turn concurred with a 9.6% to 16.3% percent increase in site sialylation. This engineering approach has thus been identified as a novel means of improving sialylation in recombinant glycoprotein therapeutics. This strategy can be utilized feasibly on its own, or in combination with existing sialylation improvement strategies. It is believed that such multi-prong approaches are required to effectively manipulate the complex sialylation process, so as to bring us closer to the goal of producing recombinant glycoproteins of high and consistent sialylation from mammalian cells. / Singapore-MIT Alliance (SMA)
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