We screened 96 stably transfected clones for the integrity of the integrated DNA using PCR, checked for single copy integrants by restriction enzyme analysis and filter hybridization and then confirmed the integrity of the attP and attB arrays by sequencing.
Therefore, we subcloned a range of our selected shRNAs into the lentiviral vector pL and stably transduced them into HIV-1 permissive HeLa P4 cells at a low multiplicity of infection (MOI: 0.1) to obtain low copy integrants and reduce the risk of insertional mutagenesis.
Importantly, both plasmidic and retroviral tagging proved to be appropriate for RMCE since they lead to a high-percentage of single copy integrants ranging from 30 to > 90% - a prerequisite for targeted integration.
Furthermore, most studies have been designed so that only cells with single copy integrants have been analysed[ 18, 19, 32, 33].
Thus, ben-1 mutants not carrying the negative selection marker, the ben-1 gene, predominantly grow and reproduce on the selection media, enabling differentiation of low copy integrants from Ex arrays and multi-copy integrants that are highly likely to have the ben-1 gene (Additional file 1, Figure S1).
We first introduced an integration target ; attP array CCAG HyTK attP array expressing the hygromycin-thymidine kinase fusion flanked by arrays of attP sites into human HT1080 cells by electroporation and, as before, selected structurally intact, single copy integrants.
We established a rescue assay based on single copy integrants of GFP-tagged CYK-4 transgenes driven by the cyk-4 promoter inserted at a defined locus in the C. elegans genome using Mos1-mediated integration (Frøkjaer-Jensen et al., 2012).
All inbred strain of mice as well as some wild mice contain multiple copies of MMTV proviral integrants, although most of them fail to produce infectious MMTV particle.
This 'passaging' procedure has been proven to yield mitotically stable integrants containing multiple copies of the transformed plasmid [ 24].
Multiple copies of the plasmid may have been integrated.
