Identification of CHO endogenous promoter elements based on a genomic library approach.
Today the standard technology for the production of many biopharmaceutical products from mammalian cell systems is frequently based on expression vectors that utilize strong mammalian active viral promoters like CMV or SV40 for driving recombinant gene transcription. On one hand these promoters allow very high expression rates, but on the other hand, they can lead to constitutive over-expression of the gene of interest resulting in a permanent stress on the cell. Another drawback is that they are cell cycle-dependent and can be subject to gene silencing which leads to a heterogeneity within the cell population. Here we aim to identify endogenous gene regulatory elements that are capable of controlling the transcription of the foreign gene. For this purpose, a genomic CHO library containing fragments of various lengths was constructed using a shotgun cloning strategy. After enzymatic fragmentation of genomic DNA, fragments encoding potential promoter regions were inserted into a promoterless vector upstream of the neomycin resistance gene. This random pool of library plasmids was transfected back into CHO cells and via cell sorting directly into 96-well plates and G418 (neomycin) selection, positive clones were isolated. A nested PCR of resistant CHO clones resulted in potential fragments which were sequenced. Putative promoter activity was predicted for these sequences by in silico methods and will be proved by re-transfection of reporter constructs into CHO cells.
Pontiller
Jens
J
Austrian Center of Biopharmaceutical Technology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Vienna, Austria. jens.pontiller@boku.ac.at
Gross
Stefan
S
Thaisuchat
Haruthai
H
Hesse
Friedemann
F
Ernst
Wolfgang
W
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D018899:CpG Islands; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000096:biosynthesis; D015698:Genomic Library; D008969:Molecular Sequence Data; D010957:Plasmids / Q000096:biosynthesis; D011401:Promoter Regions, Genetic; D050436:Regulatory Elements, Transcriptional; D012984:Software; D014162:Transfection