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Faculty Petros Tsipouras
Professor of Pediatrics
tsipouras@nso1.uchc.edu
Gene Targeting, Gene Mapping:
The development of the tetrapod limb is an intricate process which
is spatially and temporally regulated by several genes. In order to
identify genes involved in limb development and determine the role
played by these genes we have chosen several human and mouse phenotypes
which present with limb malformations. Our objective is to delineate the
gene(s) and mutations underlying a particular malformation and proceed
to test the function of these genes in a variety of experimental models.
One of the phenotypes we are currently studying is the Split Hand-Split
Foot Malformation which presents with absence of the middle rays and a
cleft in the hands and feet. A similar phenotype has been described in
the mouse where it is known as dactylaplasia. We mapped the Split
Hand-Split Foot Malformation on chromosome 10 and proceeded to narrow
the critical region containing the disease gene. An EST mapped in the
critical region appeared to have homology to a D. melanogaster gene
expressed in the imaginal disks from which the wings of the fly develop.
We used this EST to screen libraries and isolated a novel human gene
whose expression is on the embryonal ectoderm and the distal apical
ectodermal ridge. Following this approach we expect to be able both to
determine the molecular basis of human genetic disorders affecting the
limb and to identify new genes involved in the regulation of the
processes in limb development. Our collaborators in these studies are
Drs. Robert Kosher,
Caroline Dealy and other member of the Limb Development group.
Fibrillin is a large glycoprotein found in the elastin-associated
microfibrils. Microfibrils form scaffolding structures and have a
ubiquitous tissue distribution. Mutations in the fibrillin-1 gene
located on chromosome 15, FBN1, have been detected in several patients
affected with Marfan syndrome (MFS), a heritable systemic disorder of
connective tissue. The mutant fibrillin chains exercise a dominant
negative effect, that is the function of normal fibrillin molecule is
disrupted by the presence of mutant fibrillin molecules. We are
interested in determining whether the dominant negative effect of the
mutation can be ablated by selective down-regulation of the mutant FBN1
transcript. To date we have constructed several antisense hammerhead
ribozymes specific for the normal FBN1 transcript. These ribozymes are
delivered intracellularly via a receptor mediated endocytosis mechanism.
The hammerhead ribozyme is bound to a protein carrier, transferin, by
polylysine. The transferin-polylysine-plasmid complex is uptaken by cell
surface transferin receptors and the plasmid is subsequently released
from the endosomes. The effects of the ribozyme are assessed at the mRNA
level by RNase protection, and at the protein level by
immunofluorescence using fibrillin specific antibodies. Our results
showed a dramatic decrease in the amount of fibrillin synthesized by
fibroblasts as assessed by immunofluorescence. Furthermore, this effect
is specific to fibrillin and it cannot be attributed to a generalized
effect on the transcription of several genes.
This ability to specifically down-regulate cellular FBN1 mRNA and the
extracellular deposition of fibrillin suggests it might be possible to
utilize the approach to selectively down-regulate the mutant FBN1 allele
in cells derived from individuals with MFS. We have designed and
constructed a ribozyme targeted to a mutant fibrillin-1 molecule
identified in a patient with MFS and shown that this ribozyme can
selectively cleave its mutant target in cellular RNA. Delivery of this
ribozyme to cultured mutant fibroblasts will determine its ability to
ablate the dominant negative effect of the mutant FBN1 molecule and
correct the mutant cellular phenotype.
The hammerhead ribozyme might also prove to be a useful tool in the
study of the expression of endogenous genes. In particular, this
approach may provide a powerful method for the study of fibrillin and
the analysis of the role played by this and other microfibrillar
molecules in the complex process of assembly of the extracellular
microfibrils about which little is known.
Selected Publications:
Tsipouras, P., Sarfarazi, M., Devi, A., Weiffenbach, B., Boxer, M.
Marfan syndrome is closely linked to a marker on chromosome l5ql5->q2l.
Proc Natl Acad Sci USA 88:4486-4488,1991.
Tsipouras, P., Del Mastro, R., Sarfarazi, M., et al. Genetic linkage
of the Marfan syndrome, ectopia lentis, and congenital contractural
arachnodactyly to the fibrillin genes on chromosomes 15 and 5. N Engl J
Med 326:905-909,1992.
Velinov M, Slaugenhaupt SA, Stoilov 1, Scott, Jr., Cl, Gusella JF,
Tsipouras P. The gene for achondroplasia maps to the telomeric region of
chromosome 4p. Nature Genetics 6:314-317, 1994.
Kilpatrick MW, Phylactou LA, Godfrey M, Wu CH, Wu, GY, Tsipouras P.
Delivery of a hammerhead ribozyme specifically down-regulates the
production of fibrillin-1 by cultured dermal fibroblasts. Hum Molec
Genet 5:1939-1944, 1996.
Gurrieri F, Prinos P, Tackels D, Kilpatrick MW, Allanson J, Genuardi
M, Vuckov A, Nanni L, Sangiorgi E, Garofalo G, Nunes ME, Neri G,
Schwartz C, Tsipouras P. A Split Hand-Spli Foot (SHFM3) gene is located
at 10q24->25. Am J Med Genet 62:427-436, 1996.
Thomas JT, Kilpatrick MW, Lin K, Erlacher L, Lembessis P, Costa T,
Tsipouras P, Luyten FP. Disruption of human limb morphogenesis by a
dominant negative mutation in cartilage-derived morphogenetic protein-1.
Nature Genetics in press 1997. |
