Identification of novel retinoid receptors and their roles in vertebrate and invertebrate nervous systems

In vertebrates, signaling by retinoic acid (RA) is known to play an important role in embryonic development, as well as organ homeostasis in the adult. In organisms such as adult axolotls and newts, RA is also important for regeneration of the CNS, limb, tail, and many other organ systems. RA mediates many of its effects in development and regeneration through nuclear receptors, known as retinoic acid receptors (RARs) and retinoid X receptors (RXRs). This study provides evidence for an important role of the RA receptor, RAR~2, in ,( '. regeneration ofthe spinal cord and tail of the adult newt. It has previously been proposed that the ability of the nervous system to regenerate might depend on the presence or absence of this RAR~2 isoform. Here, I show for the very first time, that the regenerating spinal cord of the adult newt expresses this ~2 receptor isoform, and inhibition of retinoid signaling through this specific receptor with a selective antagonist inhibits tail and spinal cord regeneration. This provides the first evidence for a role of this receptor in this process. Another species capable of CNS ~~generation in the adult is the invertebrate, " Lymnaea stagnalis. Although RA has been detected in a small number of invertebrates (including Lymnaea), the existence and functional roles of the retinoid receptors in most invertebrate non-chordates, have not been previously studied. It has been widely believed, however, that invertebrate non-chordates only possess the RXR class of retinoid receptors, but not the RARs. In this study, a full-length RXR cDNA has been cloned, which was the first retinoid receptor to be discovered in Lymnaea. I then went on to clone the very first full-length RAR eDNA from any non-chordate, invertebrate species. The functional role of these receptors was examined, and it was shown that normal molluscan development was altered, to varying degrees, by the presence of various RXR and RAR agonists or antagonists. The resulting disruptions in embryogenesis ranged from eye and shell defects, to complete lysis of the early embryo. These studies strongly suggest an important role for both the RXR and RAR in non-chordate development. The molluscan RXR and RAR were also shown to be expressed in the adult, nonregenerating eNS, as well as in individual motor neurons regenerating in culture. More specifically, their expression displayed a non-nuclear distfibution, suggesting a possible non-genomic role for these 'nuclear' receptors. It was shown that immunoreactivity for the RXR was present in almost all regenerating growth cones, and (together with N. Farrar) it was shown that this RXR played a novel, non-genomic role in mediating growth cone turning toward retinoic acid. Immunoreactivity for the novel invertebrate RAR was also found in the regenerating growth cones, but future work will be required to determine its functional role in nerve cell regeneration. Taken together, these data provide evidence for the importance of these novel '. retinoid receptors in development and regeneration, particularly in the adult nervous system, and the conservation of their effects in mediating RA signaling from invertebrates to vertebrates.

Development of a bovine adenovirus type 2-based gene delivery vector /

ABSTRACT Recombinant adenoviruses are currently under intense investigation as potential gene delivery and gene expression vectors with applications in human and veterinary medicine. As part of our efforts to develop a bovine adenovirus type 2 (BAV2) based vector system, the nucleotide sequence of BAV2 was determined. Sixty-six open reading frames (ORFs) were found with the potential to encode polypeptides that were at least 50 amino acid (aa) residue long. Thirty-one of the BAV2 polypeptide sequences were found to share homology to already identified adenovirus proteins. The arrangement of the genes revealed that the BAV2 genomic organization closely resembles that of well-characterized human adenoviruses. In the course of this study, continuous propagation of BAV2 over many generations in cell culture resulted in the isolation of a BAV2 spontaneous mutant in which the E3 region was deleted. Restriction enzyme, sequencing and PCR analyses produced concordant results that precisely located the deletion and revealed that its size was exactly 1299 bp. The E3-deleted virus was plaque-purified and further propagated in cell culture. It appeared that the replication of such a virus lacking a portion of the E3 region was not affected, at least in cell culture. Attempts to rescue a recombinant BAV2 virus with the bacterial kanamycin resistance gene in the E3 region yielded a candidate as verified with extensive Southern blotting and PCR analyses. Attempts to purify the recombinant virus were not successful, suggesting that such recombinant BAV2 was helper-dependent. Ten clones containing full-length BAV2 genomes in a pWE15 cosmid vector were constructed. The infectivity of these constructs was tested by using different transfection methods. The BAV2 genomic clones did appear to be infectious only after extended incubation period. This may be due to limitations of various transfection methods tested, or biological differences between virus- and E. co//-derived BAV2 DNA.

The establishment of two novel bovine cell lines by transfection with the putative transforming region of bovine adenovirus type 3

Human adenoviruses (Ads), members of the family adenoviridae, are medium-sized DNA viruses which have been used as valuable research tools for the study of RNA processing, oncogenic transformation, and for the development of viral vectors for use in gene delivery and immunization technology. The left 12% of the linear Ad genollle codes for products which are necessary for the efficient replication of the virus, as well as being responsible for the forlllation of tumors in animallllodels. The establishlllent of the 293 cell line, by immortalization of human embryonic kidney cells with th~ E1 region of Ad type S (AdS), has facilitated extensive manipulation of the Ads and the development of recombinant Ad vectors. The study of bovine adenoviruses (BAVs), which cause mild respiratory and gastrointestinal infections in cattle has, on the other hand, been limited primarily to that of infectivity, immunology and clinicallllanifestations. As a result, any potential as gene delivery vehicles has not yet been realized. Continued research into the molecular biolo~gy of BAVs and the development of recolllbinant vectors would benefit from the development of a cell line analogous to that of the 293 cells. In an attelllpt to establish such a cell line, the recombinant plaslllid pKC-neo was constructed, containing the left 0-19.7% of the BAV type 3 (BAV3) genome, and the selectable marker for resistance to the aminoglycoside G418, a neomycin derivative. The plasmid construct was then used to transfect both the Madin-Darby bovine kidney (MDBK) -iicell line and primary bovine lung cells, after which G418-resistant foci were selected for analysis. Two cell lines, E61 (MDBK) and E24 (primary lung), were subsequently selected and analysed for DNA content, revealing the presence of the pKC-neo sequences in their respective genomes. In addition, BAV3 RNA transcripts were detected in the E61 cells. Although the presence of E1 products has yet to be confirmed in both cell lines, the E24 cells exhibit a phenotype characteristic of partial transformation by E1. The apparent immortalization of the primary lung cells will permit exploitation of their ability to take up exogenous DNA at high efficiency.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.