共查询到20条相似文献,搜索用时 31 毫秒
1.
Recombinant human Factor IX (rFIX) was cloned in a mammalian expression vector and transfected into CHO and HEK-293. Treatment
with 10−9 M methyl testosterone increased rFIX production by 30–50% in CHO and HEK clones. However, 10−9 M 17β-oestradiol increased production of rFIX by ~50% in CHO-F7 clone and decreased production by 48% and 37% in CHO-F8 and
HEK-F2-6, respectively. Progesterone treatment inhibited rFIX production in both cell lines. Production of rFIX can thus be
increased by sex hormone treatment and therefore used to enhance biotechnological production in mammalian cells. 相似文献
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Derouazi M Martinet D Besuchet Schmutz N Flaction R Wicht M Bertschinger M Hacker DL Beckmann JS Wurm FM 《Biochemical and biophysical research communications》2006,340(4):1069-1077
The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression. 相似文献
5.
A global RNA‐seq‐driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation 下载免费PDF全文
Jennifer D. Könitzer Markus M. Müller Germán Leparc Martin Pauers Jan Bechmann Patrick Schulz Jochen Schaub Barbara Enenkel Tobias Hildebrandt Martin Hampel Anne B. Tolstrup 《Biotechnology journal》2015,10(9):1412-1423
6.
Nan Lin Angela Davis Scott Bahr Trissa Borgschulte Katherine Achtien Kevin Kayser 《Biotechnology progress》2011,27(4):1163-1171
MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY? miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT‐PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44‐derived CHO lines producing a recombinant human IgG. We observed that miR‐221 and miR‐222 were significantly downregulated in all IgG‐producing cell lines when compared with parental DG44, whereas miR‐125b was significantly downregulated in one IgG‐producing line. In another IgG‐producing line, miR‐19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let‐7b and miR‐221 were significantly downregulated. In parental CHO K1, let‐7b, miR‐15b, and miR‐17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 相似文献
7.
To investigate the effect of human pyruvate carboxylase (hPC) on lactate formation in Chinese hamster ovary (CHO) cell lines,
FLAG-tagged hPC was introduced into a dihydrofolate-deficient CHO cell line (DG44). Three clones expressing high levels of
hPC, determined by Western blotting using an anti-FLAG monoclonal antibody, and a control cell line were established. Immunocytochemistry
revealed that a substantial amount of expressed hPC protein was localized in the mitochondria of the cells. hPC expression
did not impair cell proliferation. Rather, it improved cell viability at the end of adherent batch cultures with the serum-containing
medium probably because of reduced lactate formation. Compared with control cells, specific lactate production rate of the
three clones was decreased by 21–39%, which was because of a decreased specific glucose uptake rate and yield of lactate from
glucose. Reduced lactate formation by hPC expression was also observed in suspension fed-batch cultures using a serum-free
medium. Taken together, these results demonstrate that through the expression of the hPC enzyme, lactate formation in CHO
cell culture can be efficiently reduced. 相似文献
8.
Ciesielski MJ Kozbor D Castanaro CA Barone TA Fenstermaker RA 《Cancer immunology, immunotherapy : CII》2008,57(12):1827-1835
Survivin is a tumor-associated antigen (TAA) that has significant potential for use as a cancer vaccine target. To identify
survivin epitopes that might serve as targets for CTL-mediated, anti-tumor responses, we evaluated a series of survivin peptides
with predicted binding to mouse H2-Kb and human HLA-A*0201 antigens in peptide-loaded dendritic cell (DC) vaccines. H2-Kb-positive, C57BL/6 mice were vaccinated using syngeneic, peptide-loaded DC2.4 cells. Splenocytes from vaccinated mice were
screened by flow cytometry for binding of dimeric H2-Kb:Ig to peptide-specific CD8+ T cells. Two survivin peptides (SVN57–64 and SVN82–89) generated specific CD8+ T cells. We chose to focus on the SVN57–64 peptide because that region of the molecule is 100% homologous to human survivin. A larger peptide (SVN53–67), containing multiple class I epitopes, and a potential class II ligand, was able to elicit both CD8+ CTL and CD4+ T cell
help. We tested the SVN53–67 15-mer peptide in a therapeutic model using a peptide-loaded DC vaccine in C57BL/6 mice with survivin-expressing GL261 cerebral
gliomas. This vaccine produced significant CTL responses and helper T cell-associated cytokine production, resulting in a
significant prolongation of survival. The SVN53–67 vaccine was significantly more effective than the SVN57–64 core epitope as a cancer vaccine, emphasizing the potential benefit of incorporating multiple class I epitopes and associated
cytokine support within a single peptide. 相似文献
9.
Ling Li Jun Qin Qiang Feng Hao Tang Rong Liu Liqing Xu Zhinan Chen 《Molecular biotechnology》2011,47(1):9-17
While heparin has been shown to eliminate cell aggregation in suspension adaptations of insect and HEK293 cells for virus-based
cell cultures, the role of heparin in long period serum-free suspension adaptation of the anchorage-dependent Chinese hamster
ovary (CHO) cell lines remains inconclusive. In this paper, we explore the potential application of heparin in suspension
adaptation of CHO cell line which produces an anti-human chimeric antibody cHAb18. Heparin showed a concentration-dependent
inhibition of CHO–TS28 cell-to-cell adhesion, with a significant inhibitory effect occurring when the concentration exceeded
250 μg/ml (P < 0.001). Heparin also exhibited a cell aggregation elimination role at all concentrations (P < 0.001). Furthermore, heparin promoted cell growth and antibody secretion, with the highest cell density ((99.83 ± 12.21) × 104 cells/ml, P = 0.034) and maximum antibody yield ((9.46 ± 0.94) mg/l, P < 0.001) both occurring at 250 μg/ml heparin. When agitated, cell aggregates were effectively dispersed by 250 μg/ml heparin
and a single-cell suspension culture process was promoted. In suspension adapted CHO–TS28 cells, cell growth rates and specific
antibody productivity were maintained; while antigen-binding activity improved slightly. Together, our results show that heparin
may promote suspension adaptation of anchorage-depended CHO cells by resisting cell aggregation without reducing cell growth,
antibody secretion, and antigen-binding activity. 相似文献
10.
Cao Y Kimura S Itoi T Honda K Ohtake H Omasa T 《Biotechnology and bioengineering》2012,109(6):1357-1367
Chinese hamster ovary (CHO) cells have frequently been used in biotechnology for many years as a mammalian host cell platform for cloning and expressing genes of interest. A detailed physical chromosomal map of the CHO DG44 cell line was constructed by fluorescence in situ hybridization (FISH) imaging using randomly selected 303 BAC clones as hybridization probes (BAC-FISH). The two longest chromosomes were completely paired chromosomes; other chromosomes were partly deleted or rearranged. The end sequences of 624 BAC clones, including 287 mapped BAC clones, were analyzed and 1,119 informative BAC end sequences were obtained. Among 303 mapped BAC clones, 185 clones were used for BAC-FISH analysis of CHO K1 chromosomes and 94 clones for primary Chinese hamster lung cells. Based on this constructed physical map and end sequences, the chromosome rearrangements between CHO DG44, CHO K1, and primary Chinese hamster cells were investigated. Among 20 CHO chromosomes, eight were conserved without large rearrangement in CHO DG44, CHO K1, and primary Chinese hamster cells. This result suggested that these chromosomes were stable and essential in CHO cells and supposedly conserved in other CHO cell lines. 相似文献
11.
In Chinese hamster ovary (CHO) cells, rapid glucose metabolism normally leads to inefficient use of glucose, most of which
is converted to lactate during cell cultures. Since lactate accumulation during the culture often exerts a negative effect
on cell growth and valuable product formation, several genetic engineering approaches have been developed to suppress lactate
dehydrogenase-A (LDH-A), the enzyme converting pyruvate into lactate. However, despite the reduced lactate accumulation, such
cell cultures are eventually terminated in the late period of the culture, mainly due to apoptosis. Therefore, we developed
an apoptosis-resistant, less lactate-producing dhfr
− CHO cell line (CHO-Bcl2-LDHAsi) by overexpressing Bcl-2, one of the most well-known anti-apoptotic proteins, and by downregulating
LDH-A in a dhfr
− CHO cell line. When the dhfr
− CHO-Bcl2-LDHAsi cell line was used as a host cell line for the development of recombinant CHO (rCHO) cells producing an Fc-fusion
protein, the culture longevity of the rCHO cells was extended without any detrimental effect of genetic engineering on specific
protein productivity. Simultaneously, the specific lactate production rate and apparent yield of lactate from glucose were
reduced to 21–65% and 37–78% of the control cells, respectively. Taken together, these results show that the use of an apoptosis-resistant,
less lactate-producing dhfr
− CHO cell line as a host cell line saves the time and the effort of establishing an apoptosis-resistant, less lactate-producing
rCHO cells for producing therapeutic proteins. 相似文献
12.
Jing Tan Ting Liu Li Hou Wentong Meng Yuchun Wang Wei Zhi Li Deng 《Cytotechnology》2010,62(5):439-448
In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast
to replicate a “biomimetic niche.” The CD34+ cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2–5 weeks in the biomimetic niche (3D system)
was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2 weeks in culture,
the CD34+ cells from umbilical cord blood in the 3D system increased 3.3–4.8 folds when compared with the initial CD34+ cells. CD34+/CD38− cells accounted for 82–90% of CD34+ cells. After 5 weeks, CD34+/CD38− cells in the 3D system increased when compared with initial (1.3 ± 0.3 × 103 vs. 1.0 ± 0.5 × 104, p < 0.05), but were decreased in the 2D system (1.3 ± 0.3 × 103 vs. 2.5 ± 0.7 × 102, p < 0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6–9.3 folds vs. 1.0–1.5 folds)
after 2 weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM,
but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2–7.2 folds increase over input at 2 weeks in
culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. In conclusion, this new 3D hematopoietic
progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting
cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC
in vitro. 相似文献
13.
Simbula G Columbano A Ledda-Columbano GM Sanna L Deidda M Diana A Pibiri M 《Apoptosis : an international journal on programmed cell death》2007,12(1):113-123
α-lipoic acid (α-LA) is an antioxidant used for the treatment of a variety of diseases, including liver cirrhosis, heavy metal
poisoining, and diabetic polyneuropathy. In addition to its protective effect against oxidative stress, α-LA induces apoptosis
in different cancer cells types. However, whether α-LA acid induces apoptosis of hepatoma cells is unknown. Herein, we investigated
whether α-LA induces apoptosis in two different hepatoma cell lines FaO and HepG2. The results showed that α-LA inhibits the
growth of both cell lines as indicated by the reduction in cell number, the reduced expression of cyclin A and the increased
levels of the cyclin/CDKs inhibitors, p27Kip1 and p21Cip1. Cell cycle arrest was associated with cell loss, and DNA laddering indicative of apoptosis. Apoptosis was preceded by increased
generation of reactive oxygen species, and associated with p53 activation, increased expression of Bax, release of cytochrome
c from mitochondria, caspases activation, decreased levels of survivin, induction of pro-apoptotic signaling (i.e JNK) and
inhibition of anti-apoptotic signaling (i.e. PKB/Akt) pathways. In conclusion, this study provides evidence that α-LA induces
apoptosis in hepatoma cells, describes a possible sequence of molecular events underlying its lethal effect, and suggests
that it may prove useful in liver cancer therapy. 相似文献
14.
Production rates, abundance, chlorophyll a (Chl a) concentrations and pigment composition were measured for three size classes (<2 μm, 2–11 μm and >11 μm) of phytoplankton
from May to December 2000 in deep, mesotrophic, alpine lake Mondsee in Austria. The study focuses on differences among phytoplankton
size fractions characterised by their surface area to volume ratio ([mm2 l−1: mm3l−1]), pigment distribution patterns and photosynthetic rates. Particular attention was paid to autotrophic picophytoplankton
(APP, fraction <2 μm) since this size fraction differed significantly from the two larger size fractions. Among the three
fractions, APP showed the highest surface area to volume ratios and a high persistence in the pattern of lipophilic pigments
between temporarily and spatially successive samples (about 80% similarity of pigment composition between samples over seasons
and depths). The epilimnetic abundance of APP varied seasonally with an annual maximum of 180 × 103 cells ml−1 in June (at 4–9 m). The minimum (October at 12 m) was more than an order of magnitude lower (4.9 × 103 ml−1). APP peaked during autumn and contributed between 24% and 42% to the total area-integrated Chl a (10–23 mg m−2) and between 16% and 58% to total area-integrated production (5–64 mg m−2 h−1) throughout seasons. 相似文献
15.
The kinetics of polyethylenimine (PEI)-mediated gene transfer at early times after transfection of Chinese hamster ovary (CHO)
cell in suspension were investigated using a novel in vitro assay. Addition of an excess of competitor DNA to the culture
medium at various times after the initiation of transfection inhibited further cellular uptake of PEI–DNA particles. Using
this approach, a constant rate of particle uptake was observed during the first 60 min of transfection at a PEI:DNA ratio
of 2:1 (w/w) and a cell density of 2 × 106 cells/ml under serum-free conditions. The uptake rate declined considerably during the next 2 h of transfection. Both the
rate and the level of PEI–DNA uptake in serum-free minimal medium were found to be dependent on the PEI–DNA ratio, the cell
density at the time of transfection, and the extent of particle aggregation. These studies of the early phase of PEI-mediated
transfection are expected to lead to further opportunities for optimization of gene transfer to suspension cultures of mammalian
cells for the purpose of large-scale transient recombinant protein production. 相似文献
16.
Xiaowei Zhang Isabel Fernandez Garcia Lucia Baldi David L. Hacker Florian M. Wurm 《Biotechnology letters》2010,32(11):1587-1592
The effect of hyperosmolarity on transient recombinant protein production in Chinese hamster ovary (CHO) cells was investigated.
Addition of 90 mM NaCl to the production medium ProCHO5 increased the volumetric yield of recombinant antibody up to 4-fold
relative to transfection in ProCHO5 alone. Volumetric yields up to 50 mg l−1 were achieved in a 6 day batch culture of 3 l. In addition, hyperosmolarity reduced cell growth and increased cell size.
The addition of salt to cultures of transiently transfected CHO cells is a simple and cost-effective method to increase TGE
yields in this host. 相似文献
17.
Seyedeh Hoda Jazayeri Amir Amiri-Yekta Hamid Gourabi Baharak Abd Emami Zahra Halfinezhad Somayeh Abolghasemi Nayeralsadat Fatemi Abbas Daneshipour Mohammad Hossein Ghahremani Mohammad Hossein Sanati Mohammad Reza Khorramizadeh 《Molecular biotechnology》2017,59(11-12):490-498
Production of recombinant pharmaceutical proteins has made a great contribution to modern biotechnology. At present, quick advances in protein expression lead to the enhancement of product quantity and quality as well as reduction in timescale processing. In the current study, we assessed the expression level of recombinant human follicle-stimulating hormone (rhFSH) in adherent and suspension Chinese hamster ovary (CHO) cell lines by cultivation in serum-containing and chemically defined, protein-free media. The expression cassette entailing FSH subunits was transfected to CHO/dhfr- and CHO DG44 cell lines, and gene amplification was achieved using dihydrofolate reductase (DHFR)/methotrexate (MTX) system. Afterward, the expression level of rhFSH was studied using real-time PCR, Western blotting and ELISA. Our achievements revealed that stepwise increase in MTX [up to 2000 nano-molar (nM)] leads to boost the expression level of rhFSH mRNA in both cell lines, although DG44 have better results, as mRNA expression level reached 124.8- and 168.3-fold in alpha and beta subunits, respectively. DG44 cells have also the best protein production in 2000 nM MTX, which reached 1.7-fold in comparison with that of the mock group. According to the above results and many advantages of protein-free media, DG44 is preferable cell line for future steps. 相似文献
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Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications. 相似文献
20.
The potential of a DNA content assay, PicoGreen, for use in 3D bioengineered constructs was examined. The assay was tested
on ATDC5 cells in situ during culture in typical tissue engineering 3D constructs. Comparisons of cell standards from cell
lines and primary cells to λDNA standards was also conducted. An effective working range of the assay within 3D constructs was shown up to 2.5 × 105 cells ml−1. From significant variation found in DNA content between cell lines and primary cells, it was concluded that the most accurate
standard to use for the assay was from the cell type being examined. 相似文献