By comparing the obtained disruption targets with lists of drug molecules we find that many of these targets can be acted upon by existing drug compounds, whereas the remaining ones point at so-far unexplored targets.
For the CSRNP-1 gene disruption targeting vector, a 9.5 Kbp XhoI-ApaI genomic fragment was isolated from a BAC clone and used to construct the targeting vector.
For the CSRNP-3 gene disruption targeting vector, a 9 Kbp BamHI genomic fragment was isolated from a BAC clone and used to construct the targeting vector.
For the CSRNP-2 gene disruption targeting vector, a 2.9 Kbp HindIII-BglII upstream of exon 2 was subcloned upstream of the neo cassette, and a 5.8 Kbp XhoI containing part of exon 5 was cloned downstream, in effect creating a targeting vector, where exons 2, 3, 4 and the first 290 bp of exon 5 are deleted.
Our results also showed that lipid raft disruption targets uPAR to the lysosome in MDA-MB-231 cells.
It will be of interest to determine whether GPI disruption targets both the Shh and TGFβ pathways in HPE etiology.
Alternatively, will genes which are connecting distinct modules, and serving as key connections between integrators prove to be better disruption targets?
Since the disruption of cre2 increases the amount of cellulase activity without the severe morphological deficiencies seen with the cre1 disruption, targeting creB orthologues for disruption in other industrially useful filamentous fungi, such as A. oryzae, Trichoderma harzianum or A. niger may also prove beneficial.
To induce site specific gene disruptions, targeting endonucleases like transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases have been successfully applied to mammalian cells such as human and CHO cell lines (Galetto et al., 2009; Miller et al., 2011; Santiago et al., 2008).
Therefore, based on these results, IFD is preferable to IDM for disruption of target genes.
