Manipulating Levels of Specific MicroRNAs in Mammalian Cells - MicroRNAs as Tools in Biopharmaceutical Production - page 117

Fig. 8.1 ZFN-assisted targeted integration of a miRNA expression construct at the AAVS1 site. A
miRNA hairpin/precursor sequence plus 100-200 nucleotides upstream and downstream are cloned
after an appropriate promoter between two approximately 800 nucleotide sequences homologous
to either side of the AAVS1 integration site to generate a donor plasmid. This plasmid, along with
either a second plasmid or an RNA transcript that can express the two well-characterized ZFNs
that target the AAVS1 integration site, are transfected into cells. After the AAVS1 ZFNs make a
double-strand cut at the AAVS1 integration site, the miRNA expression construct can be integrated
by homologous recombination
where transgenes can integrate without disrupting normal cellular functions or be-
come silenced, have been identified in humans, mice, and rats. The adeno-associated
virus integration site (AAVS1) in humans (DeKelver et al. 2010 ; Lombardo et al.
2011 ) and the Rosa26 locus in mice (Casola 2010 ) are well established sites for
transgene integration and expression. Several labs have reported successful trans-
genic miRNA expression from Rosa26 in mice (Casola 2010 ; Medina et al. 2010 ),
and our own unpublished results with ZFN-technology indicate that miRNAs and
antimiRs can be targeted to and expressed from AAVS1 in human cells. Rats and
hamsters also have the Rosa26 locus, but it remains to be determined whether this site