Abstract
The production of cells capable of expressing gene(s) of interest is important for a variety of applications in biomedicine and biotechnology, including gene therapy and animal transgenesis. The ability to insert transgenes at a precise location in the genome, using site-specific recombinases such as Cre, FLP, and ΦC31, has major benefits for the efficiency of transgenesis. Recent work on integrases from ΦC31, R4, TP901-1 and Bxb1 phages demonstrated that these recombinases catalyze site-specific recombination in mammalian cells. In the present study, we examined the activities of integrases on site-specific recombination and gene expression in mammalian cells. We designed a human artificial chromosome (HAC) vector containing five recombination sites (ΦC31 attP, R4 attP, TP901-1 attP, Bxb1 attP and FRT; multi-integrase HAC vector) and de novo mammalian codon-optimized integrases. The multi-integrase HAC vector has several functions, including gene integration in a precise locus and avoiding genomic position effects; therefore, it was used as a platform to investigate integrase activities. Integrases carried out site-specific recombination at frequencies ranging from 39.3–96.8%. Additionally, we observed homogenous gene expression in 77.3–87.5% of colonies obtained using the multi-integrase HAC vector. This vector is also transferable to another cell line, and is capable of accepting genes of interest in this environment. These data suggest that integrases have high DNA recombination efficiencies in mammalian cells. The multi-integrase HAC vector enables us to produce transgene-expressing cells efficiently and create platform cell lines for gene expression.
Highlights
Many methods are available to produce transgenic cells for the functional studies of genes, drug discovery and gene therapy
Using fluorescence in situ hybridization (FISH), the digoxigenin-labeled human COT1 DNA probe localized to the MI-human artificial chromosome (HAC) vector and the HAC vector was present as an independent minichromosome without integration into the host genome in all six randomly selected clones
There were approximately 10– 460-fold more colonies observed under the platform+/recombinase+ conditions, compared with the control conditions; and approximately 6–73-fold more colonies observed under the platform+/recombinase+ conditions compared with the platform2/recombinase+ transfection. These results suggested that integrases and phage attP sites on the multi-integrase HAC (MIHAC) vector were functional in Chinese hamster ovary (CHO) cells, and that these integrases yielded higher total colony numbers compared with FLPe
Summary
Many methods are available to produce transgenic cells for the functional studies of genes, drug discovery and gene therapy. The most common method used to produce these cells relies on random integration of the gene after transfection of plasmid DNA or transduction with viruses. These methods are followed by antibiotic selection of a stable pool of cells and functional screening to identify individual clones that have the correct function(s). It is a widely held view that new gene expression technology for mammalian cells should optimally include targeting the gene to a transcriptional ‘hot spot’ in the genome [6]. Homologous recombination for targeted integration is very specific, it suffers from exceedingly low frequencies [7]
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