scholarly journals Canine and Feline Parvoviruses Can Use Human or Feline Transferrin Receptors To Bind, Enter, and Infect Cells

2001 ◽  
Vol 75 (8) ◽  
pp. 3896-3902 ◽  
Author(s):  
John S. L. Parker ◽  
William J. Murphy ◽  
Dai Wang ◽  
Stephen J. O'Brien ◽  
Colin R. Parrish
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Canine Parvovirus ◽  
Endocytic Pathway ◽  
Transferrin Receptors ◽  
Hybrid Cells ◽  
Cho Cell ◽  
Viral Capsids ◽  
Feline Panleukopenia Virus

ABSTRACT Canine parvovirus (CPV) enters and infects cells by a dynamin-dependent, clathrin-mediated endocytic pathway, and viral capsids colocalize with transferrin in perinuclear vesicles of cells shortly after entry (J. S. L. Parker and C. R. Parrish, J. Virol. 74:1919–1930, 2000). Here we report that CPV and feline panleukopenia virus (FPV), a closely related parvovirus, bind to the human and feline transferrin receptors (TfRs) and use these receptors to enter and infect cells. Capsids did not detectably bind or enter quail QT35 cells or a Chinese hamster ovary (CHO) cell-derived cell line that lacks any TfR (TRVb cells). However, capsids bound and were endocytosed into QT35 cells and CHO-derived TRVb-1 cells that expressed the human TfR. TRVb-1 cells or TRVb cells transiently expressing the feline TfR were susceptible to infection by CPV and FPV, but the parental TRVb cells were not. We screened a panel of feline-mouse hybrid cells for susceptibility to FPV infection and found that only those cells that possessed feline chromosome C2 were susceptible. The feline TfR gene (TRFC) also mapped to feline chromosome C2. These data indicate that cell susceptibility for these viruses is determined by the TfR.

Discrete electrophoretic products of mitochondrial protein synthesis in the Chinese hamster ovary cell line

1977 ◽  
Vol 55 (10) ◽  
pp. 1064-1074 ◽  
Author(s):  
R. W. Yatscoff ◽  
K. B. Freeman
Keyword(s):  
Protein Synthesis ◽  
Cell Line ◽  
Electrophoretic Mobility ◽  
Chinese Hamster Ovary ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Mitochondrial Proteins ◽  
Mitochondrial Protein Synthesis ◽  
Cho Cell ◽  
Cho Cell Line

Mitochondrial proteins labelled with [35S]methionine for 1 h in whole Chinese hamster ovary (CHO) cells in the presence of cycloheximide or emetine, known inhibitors of cytosolic protein synthesis, have been enumerated and characterized by their electrophoretic mobility in sodium dodecyl sulfate slab gel electrophoresis. Ten distinct electrophoretic bands were observed. The components were relatively stable during a 2 h postlabelling period. The same 10 bands were also seen with the CHO cell line tsH1, labelled at 40 °C, a temperature at which cytosolic but not mitochondrial protein synthesis is inhibited in this cell line, and with isolated mitochondria labelled in the presence of cycloheximide. An 11th band was present when [3H]leucine but not [35S] methionine was used for labelling. The width of the major band suggested that it consists of two components making a total of at least 12 proteins synthesized in mitochondria. The molecular weights of these mitochondrial proteins ranged from 5000 to 50000 and there was a sixfold difference in the relative molar amounts synthesized in a 1-h period in the presence of [3H]leucine or [3SS] methionine.No differences in number or electrophoretic mobility of the mitochondrially synthesized proteins were found among the seven CHO cell lines examined. These results suggest the stability of the mitochondrial genome in the CHO cell line.

scholarly journals Restoration of deficient DNA Repair Genes Mitigates Genome Instability and Increases Productivity of Chinese Hamster Ovary Cells

2021 ◽  
Author(s):  
Philipp N. Spahn ◽  
Xiaolin Zhang ◽  
Qing Hu ◽  
Nathaniel K. Hamaker ◽  
Hooman Hefzi ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Genome Instability ◽  
Therapeutic Proteins ◽  
Chinese Hamster ◽  
Dna Repair Genes ◽  
Cho Cell ◽  
Repair Genes

AbstractChinese Hamster Ovary (CHO) cells are the primary host used for manufacturing of therapeutic proteins. However, production instability of high-titer cell lines is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein titer. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide polymorphisms (SNPs) in DNA repair genes. Comparison with other mammalian cells confirmed DNA repair is compromised in CHO. Restoration of key DNA repair genes by SNP reversal or expression of intact cDNAs improved DNA repair and genome stability. Moreover, the restoration of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show for the first time that correction of key DNA repair genes yields considerable improvements in stability and protein expression in CHO, and provide new opportunities for cell line development and a more efficient and sustainable production of therapeutic proteins.

scholarly journals FACTORS AFFECTING CHINESE HAMSTER OVARY CELL PROLIFERATION AND VIABILITY

2019 ◽  
Vol 1 ◽  
pp. 245
Author(s):  
Alina Rekena ◽  
Dora Livkisa ◽  
Dagnija Loca
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cell Density ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Viable Cell ◽  
Viable Cell Density ◽  
Fed Batch ◽  
Cho Cell ◽  
Decline Phase

Advantageous cultivation procedures for the Chinese hamster ovary (CHO) cells are necessary for the productive commercial production of biopharmaceuticals. A main challenge that needs to be addressed during the process development is the differences in each cell line requirements concerning the nutrients and feed strategies in order to achieve the desired growth characteristics. Therefore, within the current research, a naïve high cell density serum free suspension adapted CHO cell line was tested with glucose and glutamine rich feeds in fed-batch Erlenmeyer shake flask cultures. Glucose consumption rate was adjusted to develop the optimal feed strategies. Obtained results indicated that high glucose and l-glutamine feeding did not improve maximum viable cell density compared to the control samples. During the exponential phase, cell proliferation and viability of all feeds showed no statistically significant difference. Instead, the fed-batch processes tested led to statistically significant differences in viable cell density and cell viability during the decline phase, compared to control (batch) culture. The difference between glucose and glutamine feeding was indistinguishable, most probably due to the concentration imbalance with the rest of the nutrients in feed. The overall study presented a method to slow down the decrease in CHO cell proliferation and viability during the decline phase, instead of increasing the maximum cell density at the plateau phase. 

scholarly journals Controlling Ratios of Plasmid-Based Double Cut Donor and CRISPR/Cas9 Components to Enhance Targeted Integration of Transgenes in Chinese Hamster Ovary Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2407
Author(s):  
Sung Wook Shin ◽  
Dongwoo Kim ◽  
Jae Seong Lee
Keyword(s):  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cho Cell ◽  
Cell Line Development ◽  
Cho Cell Line ◽  
Vector Systems ◽  
Targeted Integration

Chinese hamster ovary (CHO) cells are the most valuable expression host for the commercial production of biotherapeutics. Recent trends in recombinant CHO cell-line development have focused on the site-specific integration of transgenes encoding recombinant proteins over random integration. However, the low efficiency of homology-directed repair upon transfection of Cas9, single-guide RNA (sgRNA), and the donor template has limited its feasibility. Previously, we demonstrated that a double-cut donor (DCD) system enables highly efficient CRISPR/Cas9-mediated targeted integration (TI) in CHO cells. Here, we describe several CRISPR/Cas9 vector systems based on DCD templates using a promoter trap-based TI monitoring cell line. Among them, a multi-component (MC) system consisting of an sgRNA/DCD vector and Cas9 expression vector showed an approximate 1.5-fold increase in knock-in (KI) efficiency compared to the previous DCD system, when a systematically optimized relative ratio of sgRNA/DCD and Cas9 vector was applied. Our optimization efforts revealed that concurrently increasing sgRNA and DCD components relative to Cas9 correlated positively with KI efficiency at a single KI site. Furthermore, we explored component bottlenecks, such as effects of sgRNA components and applicability of the MC system on simultaneous double KI. Taken together, we improved the DCD vector design by tailoring plasmid constructs and relative component ratios, and this system can be widely used in the TI strategy of transgenes, particularly in CHO cell line development and engineering.

Biochemical and genetic approaches to the study of mammalian mitochondrial tRNAs

1978 ◽  
Vol 56 (6) ◽  
pp. 592-597
Author(s):  
Luke Aujame ◽  
Randall W. Yatscoff ◽  
Karl B. Freeman
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Cho Cell ◽  
Isolated Mitochondria ◽  
Slab Gels

The possible existence of mammalian mitochondrial asparaginyl-tRNA has been examined using a variety of approaches. [3H]Asparagine was incorporated into protein by mitochondria of the Chinese hamster ovary (CHO) cell line Asn-7, which has a temperature-sensitive cytosolic asparaginyl-tRNA synthetase, either in the presence of cycloheximide or at a nonpermissive temperature. Isolated mitochondria of CHO thymidine kinase minus (TK−) cells also incorporated the amino acid into protein. In each case, the number and electrophoretic mobility of the proteins was the same as mitochondrially synthesized proteins of CHO TK− ceils labelled with [35S]methionine. A tRNAAsn could be charged in isolated CHO TK cell mitochondria and the asparaginyl-tRNA was found to elute before its cytosolic counterpart on an RPC-5 column and to have a higher mobility on polyacryiamide slab gels run under denaturing conditions. This is the first demonstration of a unique mitochondrial asparaginyl-tRNA.

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