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Development and analysis of chromosome vectors
P a r t i c i p a n t s :
Cristina Auriche, Enea Gino Di Domenico, Francesca
Tilesi, post-doc fellows; Lucia Rocchi, PhD student.
Report of activity
Cystic fibrosis (CF) is a recessive congenital disease,
caused by mutations in the gene coding for CFTR
(cystic fibrosis transmembrane conductance regulator),
a chloride channel, which results in impaired
anion secretion and hyper absorption of sodium
across epithelia. Since localisation of the CFTR gene
on the long arm of chromosome 7 in 1989, a huge
effort was put into the development of gene therapy
for this life threatening disease. Because effective
therapy by in vivo delivery of therapeutic DNA
requires physiological levels of expression and longterm
maintenance, it was hypothesized that this can
be achieved by using genomic fragments that contain
all the long-range controlling elements that allow tissue-specific
gene expression at physiological level.
This is particularly important for CF since CFTR is
only expressed in specialized epithelial cells of gut,
airways, pancreas, sweat gland ducts and the male
reproductive tract. The complex pattern of expression
is dictated by regulatory elements, not fully elucidated
yet, which have been claimed to be located
upstream and downstream of the coding region and
in some introns. We have isolated for the first time to
our knowledge, the entire CFTR locus into a bacterial
DNA molecule which can be used to engineer
genomic vectors containing all the regulatory elements
necessary for spatial and temporal regulated
expression of CFTR in human epithelial cells.
The aims of the project are as follows:
1) delineate the minimal sequences required for spatial
and temporal regulated expression of CFTR in
human epithelial cells;
2) compact this information into a DNA fragment
that can be inserted into gene therapy vectors (lipid,
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Molecular genetics of eukaryotes - AREA 3
Principal investigator: Fiorentina Ascenzioni
Professor of Microbiology
Dipartimento di Biologia Cellulare e dello Sviluppo
Tel: (+39) 06 499917577, 06 49917614; Fax: (+39) 06 49917594
fiorentina.ascenzioni@uniroma1.it
cationic polymer and dendrimer- based vectors) to
achieve full levels of controlled and tissue-specific
expression of the CFTR gene;
3) Assembly of a CFTR-containing de novo chromosome
into human cell lines.
Functional analysis of the CFTR locus in
mammalian cells
The CFTR protein is involved in different activities,
the best known is the transport of the Clthrough
the membrane, other activities include
interaction with bacteria at the plasma membrane.
To asses the functional activities of the CFTR
locus cloned in the BAC vectors we have transferred
the BACs into FRT epithelial cells (Fisher
Rat Thyroid) which are routinely used to study
CFTR activity. Large scale DNA preparations from
cCFTR5A and cCFR∆12 were first analysed for
DNA integrity and subsequently used to transfect
FRT cells. Two versions of the BAC have been
used, the cCFTR5A co-transfected with pcDNAzeo
for selection, and cCFTRD12, which contains the
zeo marker. We obtained 2 cCFTR5A/clones, both
of them did not survive further growth, and 12
cCFTRD12/clones. All the cCFTR12D clones,
analysed by PCR, showed the presence of the
cCFTRD12 vector. Subsequently, CFTR mRNA
was detected by real time quantitative PCR in 7 out
of the 12 clones, although at different level.
Next, we evaluated CFTR activity by direct measurement
of electrogenic Cl- transport. The 7 clones
showing CFTR expression were seeded on
Snapwell and at 4-7 days from plating the filters
were mounted in a Ussing chamber and the
transepithelial Cl- currents were determined.
CFTR-dependent Cl- current was detected in two
clones, FRT-cl7 and FRT-cl2, the former showed
higher current with respect to the latter; three of
the clones (FRT-cl1, -cl3 and -cl4) did not show
detectable current while the remaining two (-cl5
and -cl6) showed a moderate current.