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A Mouse Transgenic Model for the P216L RDS Mutation
Digital Journal of Ophthalmology 1999
Volume 5, Number 3
June 1, 1999
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Dean Bok | Jules Stein Eye Institute and Brain Research Institute
Gabriel Travis | University of Texas Southwestern Medical Center

Animal model, Mouse, hereditary retinal degenerations
The retinal degeneration slow (rds) mouse is an interesting and useful animal model for the study of inherited retinal degenerations in humans. The murine rds gene has undergone an insertion mutation in which the second exon contains over 9 kbp of mouse genomic DNA. The inserted sequence is included in the resulting messenger RNA [1]. Accordingly the mutant message is increased in size FROM approximately 2.6 kb (wild type) to approximately 12 kb [2]. The mutant message is apparently not translated, since no protein can be detected by Western blotting [3]. Thus the rds mouse carries a null mutation.

The gene protein product of the rds wild type gene has been named rds/peripherin. The term peripherin is derived FROM the observation that this outer segment intrinsic membrane protein is distributed primarily around the perimeter of outer segment discs [4], where it appears to serve as an adhesion molecule that is essential for disc integrity [5,6]. Although fully developed inner segments and connecting cilia are formed, the homozygous rds mouse (rds-/rds-) is unable to form outer segments [7]. Heterozygous rds mice (rds+/rds-) have dysmorphic outer segments with oversized discs that often curl INTO spherical masses [8]. Thus, in rds mice, the gene is semidominant (haploinsufficiency) which means that a full complement of protein translated FROM the transcripts of both alleles is required for normal outer segment morphology.

In 1991 the first mutations were reported in the human ortholog (RDS) of murine rds/peripherin [9,10]. One of these mutations involved a codon change at position 216 FROM proline to leucine (P216L) leading to a relatively severe form of dominantly-inherited retinitis pigmentosa [9]. It them became apparent that the rds mouse could prove useful for an analysis of the cell biology and of human RDS mutations by virtue of its gene disruption. By placing the human P216L mutation on the rds +/- genetic background, we reasoned that one could realistically mimic the human disease in the mouse eye. The P216L mutation is additionally fortuitous because both the human and murine wild type protein contain proline at position 216. We have succeeded in producing several lines of transgenic mice with the P216L mutations [11] and here summarize some of the features of their photoreceptor morphology.
Materials and Methods
Generation of transgenic animals
The details of the methods are described elsewhere [11] and will be given briefly. The transgenic gene contained opsin regulatory sequences and rds cDNA followed by an SV40 sequence appropriate for the production of polyadenylated messenger RNA. This construct, in its wild type format was effective in transgenically rescuing the normal phenotype when placed on the rds-/rds- genetic background [3]. For the purpose described here, the second base on codon 216 was changed FROM "C" to "T" to encode leucine rather than proline. Fertilized oocytes FROM the murine FVBN strain were microinjected with the DNA construct and transgenic founders were crossed with C57Bl/6 mice that were wild type at rds to eliminate the rd locus which contains the mutated gene for the b- subunit of rod cGMP phosphodiesterase. The phenotype produced by the transgene was then analyzed on +/+, rds-/rds- and rds+/- genetic backgrounds. Genotype and nuclease protection analysis of endogenous and transgenic retinal RNA are described elsewhere [11].

Light and Electron Microscopy
Following deep pentobarbital anaesthesia, the animals were fixed by transcardiac perfusion of l% formaldehyde and 2% glutaraldehyde in 0.1M sodium phosphate buffer (pH 7.2). The tissues were additionally fixed in 1% osmium tetroxide dissolved in 0.1M sodium phosphate buffer (pH 7.2), dehydrated and embedded in Araldite 502 (Ciby-Geigy, Summit, NJ). Semithin and ultrathin sections were stained with toluidine blue and uranium and lead salts respectively and analyzed by light and electron microscopy.
Genotype and Levels of Transgenic messenger RNA expression
Five transgene-expressing lines of P216L animals were generated FROM the microinjections. Based on Southern blot analysis, the transgene copy number ranged FROM 1 to 10 per haploid genome and the hybridization signal indicated a single chromosomal integration site [11].

Nuclease protection analysis employing a riboprobe that protected a 169 nt fragment for endogenous rds mRNA and a 209-nt fragment for transgenic mRNA allowed us to quantitate levels of mutant mRNA expression relative to the endogenous transcripts. We were therefore able to determine that the five transgenic lines expressed the transgene in a range FROM 0.3 to 7 fold over the endogenous levels11. We were thus able to chose a line (#1376) whose endogenous and transgenic levels were essentially the same (0.5x and 0.6x respectively). Thus, on an rds+/rds- background (remember that rds- is null) line 1376 would be expected to express the rds wild type protein and rds P216L mutant protein at about equal levels. This would most closely approximate the situation in human heterozygous individuals with P216L-based dominantly inherited retinitis pigmentosa.

Light and Electron Microscopic Analysis
We analyzed the photoreceptor phenotype in three lines with an mRNA expression range of 0.3x of wild type (line #1379), 0.6x of wild type (line #1376) and 4.7x of wild type (line 1300). The photoreceptor outer segment phenotype reflected this range of expression. Figure 1 illustrates This point for the outer segments of animals at 6-7 months of age. Photoreceptors expressing 0.3x of wild type (Fig. 1A) appeared normal when compared to wild type outer segments. Conversely, photoreceptors expressing 0.6x of wild type were reduced to about 40% of their normal length (Fig. 1B) and those expressing 4.7x of wild type were reduced to about 10% or less (Fig. 1C). Surprisingly, the central feature of the phenotype was shortening of the outer segments rather than major disorganization of the discs. Outer segment discs were normally aligned except for the occasional absence or separation of the lamellar portions of cohorts of discs within the outer segment stack.

Levels of transgenic mRNA expression also correlated inversely with photoreceptor cell survival. For example, line #1300 (4.7x of wild type) had 6-7 rows of photoreceptor nuclei in the outer nuclear at one month of age, 3 rows of nuclei at 3 months and 1-2 rows at 7 months, whereas the normal number of nuclei in the outer nuclear layer was 10-ll rows in non-transgenic controls.

Phenotype of the P216L mutation on the rds heterozygous background
The nontransgenic outer segment phenotype features discs of larger than normal diameter that are curled INTO rounded masses (Fig. 2A), apparently to accommodate the larger disc diameter with the available spacing between photoreceptors. This phenotype is the result of haploinsufficiency or semidominance of the gene. Apparently, due to the functional expression of only one allele, the heterozygous rds mouse can only produce enough rds/peripherin to organize a reduced number of discs of relatively large diameter. In terms of total outer segment volume in younger heterozygotes one to two months of age, this represents about half of the volume occupied by wild type outer segments. The remaining outer segment volume is probably lost via uncontrolled budding of outer segment membrane of the type observed in homozygotes [12].

When the P216L mutation is placed on the rds+/rds- background, a slightly more severe phenotype is observed in the line whose level of transgenic mRNA expression is about equal to that of the endogenous, wild type gene (0.6x and 0.5x respectively). When age matched animals are compared, outer segments are slightly more dysmorphic than their non-trasnsgenic counterparts (Fig. 2B) . Moreover, the P216L mutation on the rds+/rds- background accelerates the rate of photoreceptor cell death over that observed in non-transgenic rds+/rds- animals. By three months of age, the animals carrying the P216L mutation have 6-7 surviving rows of photoreceptor nuclei whereas their non-transgenic, counterparts have 8-9 rows of nuclei (Table 1).
Figure 1
Electron micrographs of three different lines of rds+/rds+ mice carrying the P216L point mutation in the rds/peripherin gene. A. the level of mutant mRNA expression is 0.3x that of the endogenous, non-transgenic mRNA. Outer segments appear normal in terms of their ORDER and length. B. Mutant mRNA expression is 0.6x of the endogenous mRNA. The stacking of outer segment discs is normal, but the outer segments are only 40% of the normal length. C. Mutant mRNA expression is 4.7x of the endogenous mRNA. Outer segment discs are normally stacked but outer segments are only about 10% of normal length. The animals were 6 months (A) and 7 months (B and C) of age. X 5000.

Figure 2
Electron micrographs of photoreceptors of rds+/rds- animals that were three months old. A. Non-transgenic retina showing characteristic oversized, curled outer segment discs. B. Transgenic P216L retina in which the level of expression of endogenous mRNA coding for wild type rds/peripherin and mutant rds/peripherin is about equal. The outer segments are more dysmorphic than their nontransgenic counterparts. A microglial cell is present in the subretinal space. X 5000.

An eighteen hours old newborn presented to our casualty with prolapsed right globe outside the orbit since birth. The pupil was dilated and non-reacting to light. There was complete limitation of ocular movements with corneal exposure and severe chemosis of the conjunctiva (figure1A). Eyelids were not visible. Therefore, congenital retraction of lid and congenital ablepharon were kept as possible differentials. Lid retraction behind equator of the globe can cause proptosis, while congenital ablepharon (a very rare entity) does not lead to proptosis. Being a rapid and easily accessible orbital imaging technique NCCT was advised. NCCT of the orbit did not reveal any other abnormality (e.g. retrobulbar hematoma, teratoma, capillary hemangioma, encehalocele, meningocele, craniosynostosis). Examination under anesthesia was performed within four hours of presentation. Eyelids were seen behind the globe on gentle application of retractors. The globe was reposited by pulling the lids with forceps and simultaneously pushing the globe with a wet cotton gauze pad. A temporary suture tarsorrhaphy was done to secure the globe inside orbit. In the post-operative photograph taken at 24 hours of life one can see globe reposition with suture tarsorrhaphy (figure 1B). Tarsorrhaphy was removed after two weeks. On follow up at one month, except for a peripheral nebular corneal opacity, rest of the ocular examinations viz extraocular movements, pupillary reaction and fundus were normal (figure 1C).
Globe luxation usually follows trauma. In the present case, though there was no history of forceps application during normal vaginal delivery but still we hypothesize trauma or excessive pressure on temple region while delivery which could have led to prolapse of the globe. To the best of our knowledge, this is the first documented case of globe luxation at birth.
Supported by grants FROM the Foundation Fighting Blindness and the National Eye Institute. DB is the Dolly Green Professor of Ophthalmology.
1. Ma, J., Norton, JC., Allen AC., et al. Retinal degeneration slow (rds) in mouse results FROM the simple insertion of a repetitive genomic element INTO protein-coding exon II. Genomics 28:212-219 (1995)

2. Travis, GH., Brennan, MB., Danielson, PE., Kozak CA., Sutcliffe, JG., Identification of a photoreceptor-specific mRNA encoded by the gene responsible for retinal degeneration slow (rds). Nature 338:70-73 (1989)

3. Travis, GH., Groshan, KR., Lloyd, M., Bok, D., Complete rescue of photoreceptor dysplasia and degeneration in transgenic retinal degeneration slow (rds) mice. Neuron 9:113-119 (1992)

4. Molday, RS., Hicks, D., Molday, L., Peripherin. A rim-specific membrane protein of rod outer segment discs. Investigative ophthalmology and Visual Science 28:50-61 (1987)

5. Travis, GH., Sutcliffe, JG., Bok D., The retinal degeneration slow (rds) gene product is a photoreceptor disc membrane-associated blycoprotein. Neuron 6:61-70 (1991)

6. Connell, G., Bascom, R., Molday, L., Reid, D., McInnes, RR., Molday, RS., Photoreceptor peripherin is the normal product of the gene responsible for retinal degeneration in the rds mouse. Proceedings of the National Academy of Sciences USA 88:723-726 (1991)

7. Cohen, AI., Some cytological and initial biochemical observations on photoreceptors in retinas of rds mice. Investigative Ophthalmology and Visual Science 24:832-843 (1983)

8. Hawkins, RK., Jansen, HG., Sanyal, S., Development and degeneration of retina in rds mutant mice: Photoreceptor abnormalities in the heterozygotes. Experimental Eye Research 41:701-720 (1985)

9. Kajiwara, K., Hahn, LB., Mukai, S., Travis, GH., Berson, EL., Dryja, TP., Mutations in the human retinal degeneration slow gene in autosomal dominant retinitis pigmentosa. Nature 354:480-483 (1991)

10 Farrar, GJ., Kenna, P., Jordan, SA., et al., A three-base-pair deletion in the peripherin-RDS gene in one form of retinitis pigmentosa. Nature 354:478-480 (1991)

11. Kedzierski, W., Lloyd, L., Birch, DG., Bok, D., and Travis, GH., Generation and analysis of transgenic mice expressing P216L-substituted Rds/peripherin in rod photoreceptors. Investigative Ophthalmology and Visual Science 38:498-509 (1997)

12. Usukura, J., Bok, D., Changes in the localization and content of opsin during retinal development in the rds mutant mouse: immunocytochemistry and immunoassay. Experimental Eye Research 45:501-515 (1987)
Table 1: Comparison of outer nuclear layer (ONL) thickness in P216L Transgenic (TG) and control mice
Genotype Average Rows of Nuclei in the ONL
rds+/rds+, non-TG 10-11
P216L (4.7x wild type) on rds+/rds+
3 mo 3
7 mo 2
Non-TG rds+/rds-
2 mo 8-10
3 mo 8-9
P216L (0.6x wild type) on rds+/rds-
2 mo 6-8
3 mo 6-7