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P Pasceri

Publications and source records attributed to P Pasceri.

7 recordsLinked to original sources
PubMed1997

Evaluation of beta-globin gene therapy constructs in single copy transgenic mice.

Effective gene therapy constructs based on retrovirus or adeno-associated virus vectors will require regulatory elements that direct expression of genes transduced at single copy. Most beta-globin constructs designed for therapy of beta-thalassemias are regulated by the 5'HS2 component of the locus control region (LCR). Here we show that a human beta-globin gene flanked by two small 5'HS2 core elements or flanked by a 5'HS3 (footprints 1-3) core and a 5'HS2 core are not reproducibly expressed in single copy transgenic mice. In addition, low copy transgene concatamers that contain only dimer 5'HS2 cores fail to express, whereas those that contain monomer 5'HS2 cores express at 14% per copy. These data suggest that spacing between HS cores is crucial for LCR activity. We therefore constructed a novel 3.0 kb LCR cassette in which the 5'HS2, 5'HS3 and 5'HS4 cores are each separated by approximately 700 bp. When linked to the 815 bp beta-globin promoter this LCR directs 45% levels of expression from four independent single copy transgenes. However, the 3.0 kb LCR linked to the 265 bp promoter expresses variable levels, averaging 18%, from three single copy transgenes. Our findings suggest that sequences in the distal promoter play a role in single copy transgene activation and that larger LCR and promoter elements are most suitable for gene therapy applications.

Animals↗
PubMed1995

Mice lacking all isoforms of retinoic acid receptor beta develop normally and are susceptible to the teratogenic effects of retinoic acid.

Retinoic acids (RA) are vitamin A derivatives essential for normal embryonic development and viability of vertebrates. The RA signal is mediated by two distinct classes of receptors, RA receptors (RARs) and retinoid X receptors (RXRs). The RAR family is composed of three genes: RAR alpha, beta, and gamma. The expression of RAR beta gene is spatially and temporally restricted in certain structures in the developing embryo, suggesting that RAR beta could play specific roles during morphogenesis. Four isoforms of the RAR beta gene (beta 1-beta 4) are generated by differential usage of promoters and alternative splicing. It has recently been demonstrated that the RAR beta 2 isoform is dispensable for normal development. To ascertain the function of all RAR beta isoforms in vivo, we have generated a mutation that disrupts all isoforms of the RAR beta gene in the mouse by gene targeting in embryonic stem cells. Mice homozygous for the mutation are viable and fertile with no externally apparent abnormalities. During development, 1/11 RAR beta mutant embryos showed fusion of the ninth and tenth cranial ganglia on both sides of the hindbrain. However, no obvious alterations in the spatial pattern of expression of Hoxb-1, Hoxb-4 and Hoxb-5 were observed in day 9.5 p.c. embryos. The RAR beta null mutation did not alter the pattern or extent of the limb and craniofacial malformations induced by RA excess, suggesting that RAR beta may not be mandatory to mediate the observed teratological effects of RA in these structures. These experiments demonstrate that RAR beta isoforms are not absolutely required for embryonic development and provide additional support to the concept of functional redundancy among members of the RAR family.

Abnormalities, Drug-Induced↗
PubMed1995

Postaxial polydactyly in forelimbs of CRABP-II mutant mice.

The cytoplasmic retinoic acid (RA)-binding protein CRABP-II is expressed widely throughout early morphogenesis in mouse embryo, but its expression becomes more restricted as organogenesis progresses. CRABP-II expression remains strong in the developing limb bud suggesting a role for this protein in limb patterning. Here, we show that the CRABP-II promoter can direct expression of a lacZ transgene in a specific posterior domain during limb bud development. In order to investigate in more detail the role played by CRABP-II in RA signal transduction, we have also generated mice homozygous for a null mutation of this gene. CRABPII-/- mice are viable and fertile but show a developmental defect of the forelimb, specifically an additional, postaxial digit. This digit is generally, but not exclusively, limited to a single forepaw of an individual animal. The penetrance of the phenotype varies according to the genetic background, occurring most frequently on the inbred 129Sv background (50%), less frequently on the C57Bl/6 background (30%) and rarely on the outbred CD1 background (10%). This developmental abnormality implies a role for CRABP-II in normal patterning of the limb.

Animals↗
PubMed1989

Two UV-sensitive targets in dorsoanterior specification of frog embryos.

Previous work has shown that ultraviolet (UV) irradiation of fertilized frog eggs yields embryos that lack dorsal and anterior structures. The eggs fail to undergo the cortical/cytoplasmic rotation that specifies dorsoventral polarity, and they lack an array of parallel microtubules associated with the rotation. These eggs can be rescued by tilting with respect to gravity, and normal dorsoanterior development occurs. We find here that UV irradiation of Xenopus prophase I oocytes or Rana metaphase I oocytes also causes the dorsoanterior deficient syndrome, but the UV target is different from that in fertilized eggs. Tilting eggs, irradiated as oocytes, with respect to gravity, does not rescue dorsoanterior development, although lithium treatment does. The UV dose required to produce dorsoanterior deficiency for Rana metaphase I oocytes is much less than that for fertilized eggs, and the oocytes can form the array of parallel microtubules and undergo the cortical/cytoplasmic rotation after fertilization. Despite these features of normal development, no dorsoanterior structures form. While the UV target in fertilized eggs is thought to be the parallel microtubules (Elinson & Rowning, 1988; Devl Biol. 128, 185-197), the UV target in the oocytes may be a dorsal determinant.

Animals↗