Ocular gene therapy. From fantasy to foreseeable reality.
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Animals↗
Biomedical subjects
D J Zack
Publications and source records attributed to D J Zack.
A locus control region adjacent to the human red and green visual pigment genes.
Deletion of sequences 5' of the human red and green pigment gene array results in blue cone monochromacy, a disorder in which both red and green cone function are absent. To test whether these sequences are required for transcription of the adjacent visual pigment genes in cone photoreceptors, we produced transgenic mice carrying sequences upstream of the red and green pigment genes fused to a beta-galactosidase reporter. The patterns of transgene expression indicate that the human sequences direct expression to both long and short wave-sensitive cones in the mouse retina and that a region between 3.1 kb and 3.7 kb 5' of the red pigment gene transcription initiation site is essential for expression. Sequences within this region are highly conserved among humans, mice, and cattle, even though the latter two species have only a single visual pigment gene at this locus. These experiments suggest a model in which an interaction between the conserved 5' region and either the red or the green pigment gene promoter determines which of the two genes a given cone expresses.
Animals↗
Reconstruction of degenerate rd mouse retina by transplantation of transgenic photoreceptors.
Photoreceptors from neonatal transgenic mice with normally developing retinas were transplanted to the subretinal spaces of 2-3-month-old rd mutant mice that lack photoreceptors. The transgenic mouse photoreceptors express high levels of the lac Z reporter gene product, beta-galactosidase, which facilitated tracking the transplanted cells. Two sources were used for these cells: (1) dissection of retinal microaggregates containing photoreceptors and (2) papain-dissociated photoreceptors. Host retinas were examined after transplantation. Both methods led to survival of photoreceptors for at least 2 mo after transplantation. Relatively mature outer segments were found only in transplanted microaggregates; this occurred optimally when the cells were adjacent to the retinal pigment epithelium (RPE). beta-galactosidase-labeled outer segments associated closely with the apical processes of the host RPE, which, together with labeled phagosomes in the RPE cells, suggested functional interaction between the transplanted photoreceptors and the host RPE. This study is the first to the authors' knowledge to show electron microscopically that a morphologically normal-appearing photoreceptor layer can be reconstructed in an otherwise photoreceptorless retina.
Animals↗
Instability of immunoglobulin genes in S107 cell line.
Somatic mutation occurs frequently in rearranged and expressed immunoglobulin variable region genes in vivo. In contrast, V region hypermutation seldom occurs in antibody-forming cells in culture. The S107 mouse myeloma cell line is one of the few cell lines that has been observed to generate V region mutations frequently and spontaneously in vitro. Detailed examination reveals that both the S107 tumor and the cell line derived from it contain and express a duplicated heavy-chain gene. In culture, only one of the two heavy-chain genes undergoes both V and C region mutation, and variants with complex phenotypes and genotypes arise as a result of mutation and segregation of these duplicated genes.
Animals↗
Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas.
To define the cis-acting DNA elements required for rhodopsin expression, we generated lines of transgenic mice carrying sequences upstream of the bovine rhodopsin gene fused to the E. coli beta-galactosidase gene (lacZ). Upstream sequences extending from -2174 to +70 bp, from -734 to +70 bp, and from -222 to +70 bp direct photoreceptor-specific expression. All three -2174 lines demonstrate a superior-temporal to inferior-nasal gradient of expression across the retina, whereas lines carrying the shorter constructs demonstrate either spatially continuous expression across the retina, discrete clusters of expression, or both. As a complementary approach to defining regulatory elements, we compared DNA sequences 5' of the murine, bovine, and human rhodopsin genes. Significant homology between all three species was found just upstream of the transcription start site and at approximately 1.5 kb upstream.
Aging↗
Transplanted photoreceptors identified in dystrophic mouse retina by a transgenic reporter gene.
Dissociated photoreceptor cells from a transgenic strain of mice, containing a bovine promoter lac Z gene construct, were transplanted to the dystrophic retinas of a strain of mice (C3H, rd/rd) without obvious photoreceptors. The transgenic photoreceptor cells expressed beta-galactosidase and were distinguishable from the host retinal cells by light and electron microscopy after the 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemical reaction. These results showed that transplanted transgenic photoreceptor cells survived at least 1 month in the host retina and had a primitive outer segment and a well-developed synaptic terminal.
Animals↗
Somatic diversification of S107 from an antiphosphocholine to an anti-DNA autoantibody is due to a single base change in its heavy chain variable region.
The S107 myeloma cell line expresses the germ-line sequence of the T15 antiphosphocholine (P-Cho) antibody, which is the major antibody made by BALB/c mice in response to P-Cho, either on a variety of bacterial polysaccharides or when attached to a protein carrier. We have previously reported that a somatic mutant of the S107 cell line produces an antibody that has lost the ability to bind P-Cho and has acquired binding for double-stranded DNA. This antibody has a substitution of an alanine for a glutamic acid at residue 35 in the heavy chain variable region. We now show that this amino acid substitution is due to a single A-C transversion, which is the only nucleotide change in the heavy and light chain variable regions. Further, it appears that this change is due to somatic mutation rather than to gene conversion.
Animals↗
Monoclonal antibodies reveal the structural basis of antibody diversity.
Hybridoma technology has made it possible to introduce into continuous culture normal antibody-forming cells and to obtain large amounts of the immunoglobulin produced by each of these cells. Examination of the structure of a number of monoclonal antibodies that react with a single antigen has provided new information on the structural basis of the specificity and affinity of antibodies. Comparisons of families of monoclonal antibodies derived from a single germ line gene revealed the importance of somatic mutation in generating antibody diversity. Monoclonal antibodies that react with variable regions of other monoclonals allow the further dissection and modulation of the immune response. Finally, the continued somatic instability of immunoglobulin genes in cultured antibody-forming cells makes it possible to determine the rate of somatic mutation and to generate mutant monoclonal antibodies that may be more effective serological reagents.
Amino Acid Sequence↗
Somatically generated mouse myeloma variants synthesizing IgA half-molecules.
Whereas mouse myelomas that secrete IgA half-molecules have been shown to arise in vivo, their origin has not been definitely established. We show that somatic variants secreting phenotypically similar molecules can arise directly from the normal IgA-secreting myelomas S107 and W3082. In addition to being improperly assembled, the variant proteins have distinct carboxy-terminal deletions and an aberrant heavy-light chain disulfide bond. For at least one of the variants, variable region serology and affinity for hapten are both unaffected by these changes. Southern and Northern blot analyses indicate normal size DNA restriction fragments and mRNA, suggesting premature termination as the mechanism of deletion. These results are discussed in relation to possible mutational hot spots and long-range interdomain interactions.
Animals↗
Antigen binding variants of the S107 mouse myeloma cell line.
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Animals↗
Reporter gene expression in cones in transgenic mice carrying bovine rhodopsin promoter/lacZ transgenes.
Rhodopsin gene expression has been used as a model system to study the mechanisms regulating photoreceptor gene expression. Previous transgenic experiments using rhodopsin promoter/lacZ fusion constructs identified some of the cis-acting DNA elements responsible for photoreceptor cell-specific expression. However, the issue of rod specificity vs. photoreceptor (rod and cone) specificity of the elements was not resolved. To address this issue, the specificity of reporter gene expression in the retinas of transgenic mice carrying bovine rhodopsin promoter/lacZ (beta-galactosidase) fusion genes was assessed using X-gal staining and electron microscopy. Two independent transgenic lines, one carrying a rhodopsin promoter fragment extending from -2174 to +70 base pairs (bp) relative to the messenger RNA start site and another line carrying a fragment from -222 to +70 bp, both showed reporter gene expression in cones as well as rods, although the level of staining appeared to be less in the cones than in the rods. These results demonstrate that the -2174 to +70 bp and -222 to +70 bp bovine rhodopsin promoter fragments are not rod-specific in transgenic mice and indicate that the existence of rod promoter mediated-expression in cones must be considered when interpreting results from transgenic experiments utilizing the rhodopsin promoter.
Animals↗