Furthermore, the integrity of randomly integrated transgenes is not guaranteed, which is complicated by the fact that transgenes often integrate as concatemers containing varying numbers of transgene copies.
For example, site-specific insertion of cassettes can be carried out in preliminary randomly integrated recombinogenic sites [ 3], or the "marked" cassettes can be randomly integrated, followed by excision of the marker by a site-specific recombination system [ 1].
Presently, phiC31 integrase can perform recombination between minimal 34-bp attB and 39-bp attP sites in human cells [5] and mediates stable, site-specific integration of plasmids bearing attB into attP sites randomly integrated into the genomes of cultured human and mouse cells [6].
An important outcome of the present locus-specific integrated reporter study is highlighted by bryostatin, which the previous, randomly integrated reporter assay failed to identify as an inhibitor of Pmp22.
Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters.
This can be expected because the construct carrying the genes is randomly integrated in the genome of A. niger and its copy number varies.
Moreover, randomly integrated transgenes are often subject to pleiotropic position effects as a consequence of epigenetic changes inherent in differentiation, undermining applications in iPSCs.
Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum.
Another "Super Pgap" mutant library was further constructed where the strong native promoter Pgap of Z. mobilis was randomly integrated throughout the genome of Z. mobilis 33C via an in vitro transposon mutagenesis system.
These oligos were designed to repair a chromosomal break generated within a copy of the green fluorescent protein (GFP) gene randomly integrated into the genome of human HEK-293 cells.
Building upon these earlier attempts, in the present work we improved transformation conditions through the development of two new vectors and also gained insight into the fate of the transferred T-DNA, which was found to be randomly integrated, with a preference for repeat element-containing genome sites.
