scholarly journals Discover Stable Expression Hot Spot in Genome of Chinese Hamster Ovary Cells Using Lentivirus based Random Integration Method

2018 ◽  
Author(s):  
Songtao Zhou ◽  
Xuefeng Ding ◽  
Lei Yang ◽  
Yun Chen ◽  
Xiaohai Gong ◽  
...  
Keyword(s):  
Cell Line ◽  
Reporter Gene ◽  
Integration Method ◽  
Hot Spot ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Construction Method ◽  
Stable Expression ◽  
Random Integration ◽  
Line Construction

AbstractTraditional construction method for stable expression cell line was mainly achieved by using random integration method. However, target gene might be integrated into heterochromatin region or unstable region of chromatin by using this method, and thus required multiple rounds of selection to obtain desirable expression cell lines with stable expression level of target proteins. Rational cell line construction method can overcome this shortcoming by integrating transgenes into stable hot spot within genome specifically. Thus, to discover novel effective hot spot would be critical for this new cell line construction method. Here we reported one practical method to discover new stable hot spot by using lentivirus infection and random integration. One such hot spot located at NW_006880285.1 was thoroughly investigated in this article. The expression stability of this hot spot was verified by detecting Zsgreen1 reporter gene expression for over 50 passages. When cells were adapted to suspension culture, they successfully kept expressing Zsgreen1 reporter gene. In addition, this suspension cell line could keep expressing reporter gene stably for another 50 passages. In summary, this research offered an easy new method for researchers to identify their own stable hot spots within CHO genome, and would contribute to the development of site-specific integration study in the future.

Development of a Homogeneous MAP Kinase Reporter Gene Screen for the Identification of Agonists and Antagonists at the CXCR1 Chemokine Receptor

2001 ◽  
Vol 6 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Stephen Rees ◽  
Duncan P. Martin ◽  
Sarah V. Scott ◽  
Sue H. Brown ◽  
Neil Fraser ◽  
...  
Keyword(s):  
Map Kinase ◽  
Cell Line ◽  
G Proteins ◽  
Reporter Gene ◽  
Pertussis Toxin ◽  
Chemokine Receptor ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Receptor Activation

Agonist activity at G protein-coupled receptors (GPCRs) that regulate heterotrimeric G proteins of the Gαi/o or Gaq families has been shown to result in activation of the mitogen-activated protein (MAP) kinase cascade. To facilitate compound screening for these classes of GPCR, we have developed a reporter gene that detects the activation of the ternary complex transcription factor Sapla following MAP kinase activation. In contrast to other reporter gene assays for Gαi/o-coupled GPCRs, the MAP kinase reporter generates an increase in signal in the presence of agonist. The reporter gene has been transfected into Chinese hamster ovary cells to generate a "host" reporter gene-containing cell line. The Gαi-coupled human CXCR1 chemokine receptor was subsequently transfected into this cell line in order to develop a 384-well format screen for both agonists and antagonists of this receptor. Agonists activated the reporter gene with the expected rank order of potency and with similar concentration dependence as seen with the regulation of other signal transduction cascades in mammalian cells: interleukin-8 (IL-8) (pEC50 = 7.0 ± 0.1) > GCP-2 (pEC50 = 6.3 ± 0.1) > NAP-2 (pEC50 < 6). CXCR1-mediated activation of MAP kinase was inhibited by pertussis toxin and the MEK inhibitor PD98059, demonstrating that receptor activation of MAP kinase is due to pertussis toxin-sensitive Gα/i/o-family G proteins to cause the activation of MEK kinase. Using the 384-well format, assay performance was unaffected by solvent concentrations of 0.5% ethanol, 0.15% glycerol, or 1% DMSO. Signal crosstalk between adjacent wells was less than 1%. The assay exhibited a Z factor of 0.53 and a coefficient of variation of response to repeated application of IL-8 (100 nM) of 15.9%.

scholarly journals Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells.

1982 ◽  
Vol 2 (3) ◽  
pp. 250-257 ◽  
Author(s):  
J A Tischfield ◽  
J J Trill ◽  
Y I Lee ◽  
K Coy ◽  
M W Taylor
Keyword(s):  
Chinese Hamster Ovary ◽  
Hot Spot ◽  
Chinese Hamster Ovary Cells ◽  
Human Fibroblasts ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Adenine Phosphoribosyltransferase ◽  
Ovary Cells ◽  
L Cells ◽  
A Site

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.

Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells

1982 ◽  
Vol 2 (3) ◽  
pp. 250-257
Author(s):  
J A Tischfield ◽  
J J Trill ◽  
Y I Lee ◽  
K Coy ◽  
M W Taylor
Keyword(s):  
Chinese Hamster Ovary ◽  
Hot Spot ◽  
Chinese Hamster Ovary Cells ◽  
Human Fibroblasts ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Adenine Phosphoribosyltransferase ◽  
Ovary Cells ◽  
L Cells ◽  
A Site

Resistance to adenine analogs such as 2,6-diaminopurine occurs at a rate of approximately 10(-3) per cell per generation in mouse L cells. This resistance is associated with a loss of detectable adenine phosphoribosyltransferase activity. Other genetic loci in L cells have the expected mutation frequency (approximately 10(-6)). Transformation of L cell mutants with Chinese hamster ovary cell DNA results in transformants with adenine phosphoribosyltransferase activity characteristic of Chinese hamster ovary cells. No activation of the mouse gene occurs on hybridization with human fibroblasts. That this high frequency event is the result of mutation rather than an epigenetic event is supported by antigenic and reversion studies of the 2,6-diaminopurine-resistant clones. These results are consistent with either a mutational hot-spot, a locus specific mutator gene, or a site of integration of an insertion sequence.

scholarly journals Enhancement of cytotoxicities of ricin and Pseudomonas toxin in Chinese hamster ovary cells by nigericin.

1981 ◽  
Vol 1 (6) ◽  
pp. 552-559 ◽  
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein Synthesis ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Diphtheria Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Pseudomonas Aeruginosa Exotoxin

Nigericin and monensin, ionophores for Na+ and K+, have been found to enhance the cytotoxicities of abrin, ricin, and Pseudomonas aeruginosa exotoxin A in Chinese hamster ovary (CHO) cells. They do not affect the cytotoxicity of diphtheria toxin in the same cell line. Maximal sensitization of the CHO cells toward ricin and Pseudomonas toxin requires preculture of CHO cells in the presence of nigericin. Inhibition of protein synthesis in CHO cells by ricin or Pseudomonas toxin is also enhanced by preculture of CHO cells in the presence of nigericin. These results suggest a common step in the intoxication process of ricin and Pseudomonas toxin, the rate of which is facilitated by pretreatment with nigericin. This step is, however, not shared by the intoxication of CHO cells with diphtheria toxin.

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