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F. Ascenzioni - Development and analysis of chromosome vectors<br />
The different amount of the CFTR mRNA and the<br />
different activity of the Cl- channel observed<br />
among the FRT clones analysed, could be due to<br />
differences in the copy number of the integrated<br />
locus and/or to epigenetic effects due to the nearby<br />
chromatin. To elucidate this point we determined<br />
the copy number of the human CFTR locus in the<br />
two most active clones, FRT-cl2 and FRT–cl7. By<br />
real time PCR we detected two copies of the human<br />
CFTR locus in FRT-cl7 and one copy in CFTR-cl2<br />
cells. FRT-cl7 cells were further analysed by FISH<br />
which could detect only one integration site into a<br />
big host chromosome, suggesting that both copies<br />
of the locus become integrated in the same site of<br />
the host genome.<br />
Finally the CFTR protein was detected by immunofluorescence<br />
analysis in FRT-cl7, which showed the<br />
highest CFTR mRNA level and Cl- current. For this<br />
purpose FRTcl7 cells were seeded in transwell (Costar)<br />
and let polarized for 5-7 days until the transepithelial<br />
resistance was in the range 1-2 KΩ cm 2 . CFTR protein<br />
was immunolocalized by anti-CFTR antibody and confocal<br />
microscopy. The result of this analysis showed<br />
the classical honey-bee distribution of the CFTR in<br />
the xy plane, and compartimentalization above the<br />
nucleus in the xz plane, strongly suggesting that the<br />
CFTR protein was properly transported to the plasmamembrane<br />
in FRT-cl7 cells.<br />
In conclusion these results clearly demonstrated that<br />
the human CFTR locus isolated in the BAC molecule<br />
is fully functional and provide good material for the<br />
remaining aims of the project.<br />
De novo chromosome assembly with the<br />
human CFTR locus<br />
One possibility to complement a genetic defect by<br />
using an entire locus is de novo assembly of an arti-<br />
30<br />
ficial chromosomes containing the gene of interest.<br />
We have performed preliminary experiments to<br />
determine the feasibility of this approach using the<br />
cCFTR5A BAC as a donor of the CFTR gene. For<br />
this purpose cCFTR5A and alphoid-PAC DNAs<br />
were co-transfected in HT1080 cells and after<br />
selection for markers present in the alphoid construct<br />
we have identified, among the positive clones,<br />
three clones that contained also the cCFTR5A construct.<br />
One of these clones showed the presence of<br />
a de novo artificial chromosomes as revealed by<br />
FISH with CFTR and alphoid probes. This result<br />
provided proof of principle for delivery into mammalian<br />
cells of the CFTR locus by de novo chromosome<br />
assembly.<br />
Selected publications<br />
Conese M, Boyd AC, Di Gioia S, Auriche C,<br />
Ascenzioni F. Genomic context vectors and artificial<br />
chromosomes for cystic fibrosis gene therapy. Curr<br />
Gene Ther. 2007, 7:175-87.<br />
Di Domenico EG, Auriche C, Viscardi V, Longhese<br />
MP, Gilson E, Ascenzioni F. The Mec1p and Tel1p<br />
checkpoint kinases allow humanized yeast to tolerate<br />
chronic telomere dysfunctions by suppressing telomere<br />
fusions. DNA Rep. 2008, Epub.<br />
Pirone L, Bragonzi A, Farcomeni A, Paroni M,<br />
Auriche C, Conese M, Chiarini L, Dalmastri C,<br />
Bevivino AM, Ascenzioni F. Burkholderia cenocepacia<br />
strains isolated from cystic fibrosis patients are<br />
apparently more invasive and more virulent than<br />
rhizosphere strains. Environ Microbiol. 2008,<br />
10:2773-84.