Discovery of a stable expression hot spot in the genome of Chinese hamster ovary cells using lentivirus-based random integration

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.

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Stable expression of human α-2,6-sialyltransferase in Chinese hamster ovary cells: functional consequences for human erythropoietin expression and bioactivity

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/s0304-4165(98)00095-6 ◽

1998 ◽

Vol 1425 (3) ◽

pp. 441-452 ◽

Cited By ~ 33

Author(s):

Xiaoqian Zhang ◽

Serene H.L. Lok ◽

Oi Lian Kon

Keyword(s):

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Stable Expression ◽

Ovary Cells ◽

Human Erythropoietin ◽

Functional Consequences

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

10.1101/467936 ◽

2018 ◽

Cited By ~ 1

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.

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Genetic instability at the adenine phosphoribosyltransferase locus in mouse L cells

Molecular and Cellular Biology ◽

10.1128/mcb.2.3.250-257.1982 ◽

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.

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