Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-11-14
2004-06-15
Helms, Larry R. (Department: 1642)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C536S023530
Reexamination Certificate
active
06750041
ABSTRACT:
BACKGROUND
Recombinant expression constructs are routinely used to generate stable mammalian cell lines expressing a desired recombinant protein. These recombinant expression constructs may be of a type which remain as extrachromasomal plasmids in a transfected host cell, or may be a type which integrates into the genome of the host. Mammalian host cells which carry stably integrated copies of recombinant genes are typically more reliable with respect to maintenance and integrity of the recombinant gene. Once a recombinant mammalian host cell is identified which produces the recombinant protein, integrated copies of the recombinant gene may be more desirable to extrachromasomal copies of the gene.
However, the frequency of cell lines carrying stably integrated copies of a recombinant gene that express a desired recombinant protein at high levels is quite low. Typically, large numbers of stably transfected mammalian cells must be screened to identify clones which express the recombinant protein at high levels. This is widely believed to be due to the effects of the locus of insertion of the recombinant gene into the mammalian genome. Due to the size of the mammalian genome it is highly unlikely that a random integration event would result in the insertion of the recombinant gene into a locus favorable for high levels of gene expression.
An increase in the frequency of high level recombinant gene expressing cell lines would provide a much greater pool of high expressors to choose from for subsequent selection as the recombinant protein producer. In addition, the frequency increase would reduce the number of stable cell lines that would need to be generated to identify high level recombinant protein expressors.
SUMMARY OF THE DISCLOSURE
This invention discloses a method of defining a specific position in the mammalian genome that is favorable for high levels of recombinant gene expression and, an efficient method for increasing the frequency of high expressors by targeting the expression construct to a favorable genomic position.
Also disclosed is the operation of the method of this invention to establish a stable cell line expressing high levels of a recombinant protein. Further the invention shows that the cell line generated using this invention, expresses recombinant protein at extremely high levels, frequently exceeding the best expressing cell lines produced by random integration of the expression construct into the mammalian genome.
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Hollis Gregory Franklin
Mark George E.
Heber Sheldon O.
Helms Larry R.
Merck & Co. , Inc.
Tribble Jack L.
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