The use of a stringent selection system allows the identification of DNA elements that augment gene expression.
The use of high stringency selection systems often results in the induction of very few recombinant mammalian cell lines, which limits the ability to isolate a cell line with favorable characteristics. The employment of for instance STAR elements in DNA constructs elevates the induced number of colonies and also the protein expression levels in these colonies. Here, we describe a method to systematically identify genomic DNA elements that are able to induce many stably transfected mammalian cell lines. We isolated genomic DNA fragments upstream from the human Rb1 and p73 gene loci and cloned them around an expression cassette that contains a very stringent selection marker. Due to the stringency of the selection marker, hardly any colony survives without flanking DNA elements. We tested fourteen ~3500 bp DNA stretches from the Rb1 and p73 loci. Only two ~3500 bp long DNA fragments, called Rb1E and Rb1F, induced many colonies in the context of the stringent selection system and these colonies displayed high protein expression levels. Functional analysis showed that the Rb1 DNA fragments contained no enhancer, promoter, or STAR activity. Our data show the potential of a methodology to identify novel gene expression augmenting DNA elements in an unbiased manner.
Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098XH Amsterdam, The Netherlands.
Femke
F
Hoeksema
Rik
R
van Blokland
Michel
M
Siep
Karien
K
Hamer
Tjalling
T
Siersma
Jan
J
den Blaauwen
John
J
Verhees
Arie P
AP
Otte
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016160:Retinoblastoma Protein / Q000235:genetics*; D014162:Transfection
D016160:Retinoblastoma Protein