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