Biomedical subjects

Teri Seeberger

Publications and source records attributed to Teri Seeberger.

4 recordsLinked to original sources

PubMed2004

Characterization of a mutation in the lens-specific CP49 in the 129 strain of mouse.

PURPOSE: The 129 strain of mouse carries a mutation in the gene for CP49 (phakinin), an intermediate filament protein thus far demonstrated only in the lens fiber cell. As such, these mice represent naturally occurring mutants of interest in the study of the lens cytoskeleton. However, this strain of mouse is also widely used as a source of embryonic stem cells in gene-targeting studies. The presence of a mutation in a lens-specific gene can confound interpretation of studies in which lens genes have been knocked out. In the present study, both the genotype and phenotype of these mice were characterized, to permit an evaluation of the biological impact of this mutation and to facilitate the discrimination between wild-type and mutant animals that have been derived from this strain in gene-targeting studies. METHODS: The CP49 cDNA and, when relevant, the genomic DNA sequences were determined for the 129/SvJ and C57BL/6J mice and from a commercially available 129/OLa P1 genomic clone. PCR primers were screened for their capacity to discriminate between the mutant and wild-type CP49 genes. Northern blot analysis was used to assess mRNA levels for CP49, filensin, and gammaS-crystallin (control). Western blot analysis was used to identify changes in protein size and abundance. The impact of the mutation on lens architecture was evaluated at the light-microscope level. Lens fiber cell ghosts from mutant and wild-type mice were examined in the electron microscope for the presence of beaded filaments. Lens clarity was assessed by slit lamp. RESULTS: The 129 strain of mice exhibited a 6303-bp deletion from the end of intron B, and the beginning of exon 2. This deletion results in the loss of the exon 2 splice acceptor site, absence of exon 2 from the CP49 mRNA, and dramatically reduced levels of CP49 mRNA. The CP49 protein was undetectable by Western blot analysis. Messenger RNA levels for filensin, CP49's assembly partner, were normal, but protein levels were sharply reduced. Light microscopy established that the initial differentiation and elongation of the fiber cells proceeded normally. Electron microscopy showed the absence of beaded filaments, whereas slit lamp microscopy showed a slowly emerging and progressive loss of optical clarity. CONCLUSIONS: The 129/SvJ and 129/OLa strains of mice harbor a mutation that sharply reduces CP49 mRNA levels and essentially eliminates both CP49 and the beaded filament. These lenses exhibited a slow but progressive loss of optical clarity with age. Thus, the 129 strain of mouse behaves as a functional CP49 knockout. The loss of clarity in the lenses of these animals and the absence of beaded filaments (and any attendant interactions that may exist between beaded filaments and other lens proteins/structures) suggest that gene-targeting studies of lens proteins in which the 129 strain was used as a source of embryonic stem cells may need reevaluation.

Amino Acid Sequence↗

PubMed2003

Targeted deletion of the lens fiber cell-specific intermediate filament protein filensin.

PURPOSE: To determine the function of the lens fiber cell-specific cytoskeletal protein, filensin, in lens biology. METHODS: Targeted genomic deletion was used to delete exon 1 and the transcriptional start site of the filensin gene. Resultant chimeric animals were bred to homozygosity for the mutant allele. These animals were outbred to mice bearing the wild-type CP49 alleles to eliminate the mutant CP49 gene carried by the 129 strain of mice. Animals homozygous for the mutated filensin gene and wild-type CP49 gene were compared with wild-type and heterozygous animals by Northern and Western blot analyses, light and electron microscopy, and slit lamp microscopy. RESULTS: Disruption of the filensin gene successfully blocked production of filensin mRNA, reduced levels of filensin's assembly partner CP49, and prevented the assembly of beaded filaments. Despite the absence of beaded filaments, lenses did not show obvious changes in fetal development, nor in the differentiation of epithelial cells into mature fiber cells, as judged by light microscopic analysis. Filensin knockouts began to show evidence of light-scattering by 2 months and worsened with age. Heterozygous animals exhibited an intermediate phenotype, showing a reduction in filensin transcript and moderate light-scattering at 5 months. CONCLUSIONS: The lens fiber cell-specific intermediate filament protein filensin is essential for beaded filament assembly. However, although beaded filaments are not needed for normal lens fetal development or fiber cell differentiation, they appear to be necessary for the long-term maintenance of optical clarity. The mechanism by which the absence of filensin and the beaded filament affects optical clarity has yet to be defined.

Animals↗

PubMed2002

Non-visual information does not drive saccade gain adaptation in monkeys.

Recent experiments have characterized the dependence of saccade gain adaptation on the characteristics of the visual error following inaccurate saccades. We currently know little about the potential role of non-visual information in driving saccade adaptation. The brain could use non-visual signals from the saccade burst generator or extraocular muscle (EOM) proprioceptors to determine if the eye had rotated the appropriate distance to aim at a target. Both saccade-related burst signals and EOM proprioceptive information reach the posterior vermis of the cerebellum, a brain area strongly implicated in saccade adaptation. In the experiment described here we determined if non-visual information has a significant affect on saccade adaptation. We made monkey saccades hypometric with intra-saccade target movements and then tested the recovery of saccade gain toward normal under three conditions: (1) when the target was continuously visible, (2) when the target extinguished for 1000 ms beginning during the saccade, and (3) when the monkey remained in the dark. In the first condition both visual and non-visual indications of hypometria were available. In the second, only non-visual information was available. In the third, the monkey made no visually guided saccades and very few spontaneous saccades in the dark so neither visual nor non-visual information could drive adaptation. We found that, though it was hypometric, saccade size during recovery changed the same small amount when monkeys made saccades to extinguishing targets or remained in the dark. Saccade size changed significantly (approximately 5x) more during recovery when the monkey tracked continuously visible targets. Thus non-visual information has no influence on adaptation and visual post-saccade error is the only known driver of saccade adaptation.

Adaptation, Physiological↗

PubMed2002

Targeted genomic deletion of the lens-specific intermediate filament protein CP49.

PURPOSE: To deduce the function of the lens-specific cytoskeletal structure, the beaded filament, by blocking expression of the fiber cell-specific beaded filament protein CP49. METHODS: The first exon of the mouse CP49 gene was deleted by using targeted genomic deletion techniques. Gene deletion was assessed through Southern blot analysis and PCR. Translation and protein expression were characterized by Northern and Western blot analysis of both CP49 and its assembly partner filensin. The architecture of knockout lenses was compared with that of wild-type lenses at the histologic level by light microscopy. Lens clarity was assessed in situ by direct ophthalmic examination and slit lamp microscopy. RESULTS: Transcription and translation of CP49 were successfully negated in knockout animals. Lenses homozygous for the CP49 deletion showed no obvious changes in lens architecture at the light microscope level. Filensin levels were sharply reduced, although filensin mRNA levels appeared unchanged. Direct examination of lenses showed no obvious loss of lens clarity, but slit lamp examination revealed the emergence of opacification in even the youngest animals. The opacification worsened with age. CONCLUSIONS: The absence of CP49 causes a subtle loss of optical clarity in the ocular lens, a loss that worsens with age. However, CP49 is not essential for the assumption or maintenance of overall fiber cell shape or long-range order of fiber cells. CP49 appears to regulate the protein levels of its assembly partner filensin, suggesting a mechanism for the regulation of beaded filament protein stoichiometry.

Aging↗