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The goal of the research is to examine which DNA elements lead to the highest and most continuous
protein expression levels of transgenes in mammalian cells. The research has both practical and
theoretical facets. The practical part is to obtain recombinant mammalian cell lines with continuous
and high protein expression levels. This is a challenge, due to the usually instable and low protein
production levels of recombinant genes. To reach this, several DNA vectors were constructed using a
selection of combinations of DNA elements, previously identified as gene expression enhancers.
Herein lays the theoretical aim: examination of which DNA elements lead to the highest and most
continuous protein expression levels of transgenes in mammalian cells, in this case Chinese hamster
ovary (CHO) cells. Mutants of several of these vectors are made using PCR (poly chain reaction) to
modify one of the gene expression enhancer elements. The mutants are used to investigate which
sequences of this DNA element are vital for yielding high levels of protein of the transgene, here the
reporter gene GFP (green fluorescent protein). This gene is used because the associated protein can
be easily measured in the cell, using flowcytometer (FACS). To create mammalian cell lines with a
continuous and high expression of a transgene, a stringent selection system is used. This is done by
constructing the DNA vectors in such way that the transcription of the reporter gene (e.g. GFP, an
antibody) is coupled to the transcription of a gene necessary for the survival of the cell, the marker
gene (e.g. an antibiotic resistance gene). In addition to a selection of DNA elements, a study is
performed applying four different selection marker genes, including the wild type. The marker genes
respond to the same antibiotic, but differ in the ease at which they are transcribed. Hence, the DNA
vectors differ in the use of DNA elements as well as the employment of variants of the marker gene.
As a result, if the cell produces a large amount of the antibiotic resistance protein in the presence of
the antibiotic, simultaneously high levels of the reporter protein are attained. The heterologous
promoter used, is the human β-actin promoter. Therefore, the stringency of the system can be
modified by altering the ease of which the marker gene can be transcribed.