[34] Animal models of inherited retinal diseases

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[34] Animal Models of Inherited Retinal Diseases By

J E A N N E F R E D E R I C K , J. D A R I N B R O N S O N , a n d W O L F G A N G B A E H R

Introduction: Genes Linked to Retinitis Pigmentosa and Allied Diseases Of several hundred inherited diseases affecting the human eye or the retina, relatively few have been characterized at a molecular genetic level. Among these, retinitis pigmentosa (RP), a large and heterogeneous group of blinding disorders, has been studied intensively at the molecular level. 1'2 Particularly useful in determining biochemical mechanisms has been animal models of hereditary degeneration that simulate human diseases. In this chapter, we summarize vertebrate animal models of retinal degeneration consisting of two groups: the naturally occurring, and the laboratory generated. Naturally occurring mutations affect a variety of genes, including genes involved in phototransduction (e.g., rd mouse, rd chicken), genes encoding structural proteins of photoreceptors (e.g., rds mouse), genes encoding transcription factors (e.g., vitiligo), or genes also expressed in other tissues (e.g., shaker mouse). Laboratory-generated mutants consist typically of transgenic mice or mice in which a gene of interest has been knocked out. Other vertebrate animals, for example, Xenopus and zebrafish, are used increasingly to generate animal models. Genes targeted include those involved in photoreception (rhodopsin), phototransduction (cGMP cascade), the visual cycle, cellular metabolism (transporters, channels, enzymes), cell structure (cytoskeleton, membrane components), or genes involved in development (transcription factors). Naturally OccuiTing Animal Models of Retinitis Pigmentosa Several models of both categories have defects in genes encoding components of the phototransduction cascade (PC) (Fig. 13,4) or the visual cycle (VC). We estimate the number of genes involved in these events conservatively at about 40-60. About two dozen have been cloned and characterized by recombinant DNA techniques (Fig. 1). The number of identified PC/VC gene defects producing naturally occurring animal models 1 T. 2 K. 3 A. 4 y.

P. Dryja and T. Li, Hum. Mol. Genet. 4, 1739 (1995). G r e g o r y - E v a n s and S. S. Bhattacharya, Trends Genet. 14, 103 (1998). Polans, W. Baehr, and K. Palczewski, Trends. Neurosci. 19, 547 (1996). Koutalos a n d K. W. Yau, Trends Neurosci. 19, 73 (1996).

METHODS IN ENZYMOLOGY,VOL. 316

Copyright © 2000by AcademicPress All rights of reproduction in any form reserved. 0076-6879/00$30.00

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of RP is surprisingly small (four). These genes encode PDE~ (rd or r mouse, rcdl Irish setter), PDE~ (rcd3 Cardigan Welsh dog), GC1 (rd chicken), and RPE65 (Briard dog). PDE (cGMP phosphodiesterase) is the enzyme that degrades cGMP on (indirect) activation by light, and guanylate cyclase 1 (GC1) is the enzyme that produces cGMP (Fig. 1). Because cytoplasmic cGMP levels in dark and light are carefully balanced by a number of feedback mechanisms, it is perhaps not surprising that defects in genes encoding these enzymes cause severe problems in photoreceptor metabolism. RPE65 is a component of the retinal pigment epithelium (RPE) involved in retinoid metabolism. The number of naturally occurring animal models with known gene defects affecting photoreceptor structure is even smaller (one, rds mouse). A number of animal models (e.g., RCS rat, prcd dog) have been researched thoroughly but the causative gene defects remain unknown. Laboratory-Generated Animal Models Owing to rapidly advancing transgenic and gene-targeting technologies, the number of laboratory-generated vertebrate animals is ever increasing (Table I). Mutations in the rhodopsin gene, responsible for a large number of dominant and recessive RP cases in human, have been used most extensively for the generation of RP models. 5 A number of genes that have been identified in photoreceptors or the inner retina have been used for generation of knockout animals, a powerful method by which to analyze the contribution of a gene product to a specific pathway. Hence the majority of animal models listed in Table I are knockout models. 5 j. Lem and C. L. Makino, Curt. Opin. Neurobiol. 6, 453 (1996).

F1G. 1. The phototransduction cascade and animal models for retinal degeneration. The cascade 3 consists of rhodopsin (R, one subunit), transducin (T, three subunits), cGMP phosphodiesterase (PDE, four subunits), and the cGMP-gated cation channel (two subunits). The task of the cascade is to rapidly hydrolyze cytoplasmic cGMP and to close CNG cation channels. Regulatory components are rhodopsin kinase (RK), arrestin (Art), recoverin (Rec), phosducin (Pdc), RGS9 (GAP), and calmodulin (CAM), each of which consists of one subunit. The recovery phase returns activated components and cGMP/Ca 2÷ levels to their dark states. Key components of the recovery phase are guanylate cyclase (GC; two distinct GCs have been identified in photoreceptors) and guanylate cyclase-activating proteins (GCAPs; three identified to date). The cation exchanger (CaX, one subunit) is not part of the cascade but is instrumental in regulating cation concentrations. Arrows point to subunit genes whose defects have been identified in naturally occurring animal models, or to genes that have been used for targeting in knockout animal models. For reviews see Polans et aL 3 and Koutalos and Yau. 4

518

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TABLE I VERTEBRATE ANIMAL MODELS (EXCLUDING HUMAN), GENE DEFECTS, AND PHENOTYPES a

Component/gene b Rhodopsin (rod)

Transducin (rod) Transducin (cone) cGMP phosphodiesterase (PDE)

Rho

Gt~

PDE.

Activity/functionb Light reception; catalyst of Gt~" GTP formation; disk structural protein

Activator of rod PDE Activator of cone PDE Hydrolyzes cGMP

PDE~

PDE~ Guanylate cyclase 1 Guanylate cyclase-activating proteins 1 and 2 Recoverin (Smodulin) Arrestin (rod) Rho-kinase (rod and cone) IRBP (rod and cone) RPE65

GC1

Produces cGMP

GCAP-1, -2

Mediate Ca 2÷ sensitivity of GC1 and or GC2 Ca 2+ sensor; blocks Rho kinase? Binds to phosphorylated Rho Phosphorylates Rho Retinoid-binding protein

A B C R (rim protein)

Transporter

rds/peripherin ROM1 Mitf

Structural Structural Transcription factor ? ?

? ?

Animal model c Transgenic mouse

Transgenic pig Knockout mouse Knockout mouse

Gene defect d P23H V20G; P23H; P27L T17M P347S Q344ter G90D K296E P347L

Transgeic mouse

rcd3 cardigan Welsh rd mouse r mouse rcdl Irish setter Knockout mouse Transgenic mouse rd chicken Knockout mouse Double knockout mouse

Phenotype Dominant RP Dominant RP

CSNB

Dominant RP Recessive RP

Downregulation of PDE Exon 15 frameshift Y348ter Y348ter W807ter Null allele

Refs. e 1 2 3 4-6 7 8, 9 10 11, 12 13-15 16 17 18

Recessive RP Recessive RP Recessive RP

19 20 21-22 23 24 25 26 27

A × 4-7 Null allele Null allele

Recessive LCA I

Knockout mouse

Null allele

None

28

Knockout mouse

Null allele

Oguchi CSNB

29, 30

Knockout mouse?

31

Knockout mouse

Null allele

CSNB Briard dog Knockout mouse Knockout mouse

4-bp deletion

rds mouse

Insertion Null allele Defect in mitf gene ? ?

Knockout mouse vitiligo mouse RCS rat

Rdy cat

Null allele

32

LCA type II Recessive Stargardt MD Recessive RP Recessive RP

ad rod-cone dysplasia

33, 34 35, 36 37

38, 39 40 41, 42 43, 44 45

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TABLE I (continued)

Component/gene b

Activity/functionb

Animal model C

Gene defect d

Phosducin?

Regulatory protein

prcd

? mGluR6

? Metabotropic Glu receptor, type 6 ?

Rcd2 Knockout mouse

? /33 subunit gene of AP3 adaptor complex

?

nob mouse pearl mouse

?

REP-1

Knockout mouse

Null allele

Myosin VIIA

Rab escort protein Structural

shaker-1 mouse

Multiple mutations

ND PLC/34

Phospholipase

Knockout mouse Knockout mouse

Null allele Null allele

Knockout mouse

Null allele

?

Cyclin D1

Phenotype

Missense mutation R82G? Null allele

Mutant fish (nba)

OAT

Ornithine & amino transferase

Knockout mouse

Null allele

Dystrophin dp260

?

Knockout mouse

Null allele

Refs. e 46, 47

Recessive RP Absence of bwave Dominant night blindness xCSNB Night blindness HermanskyPudlak syndrome Choroideremia Usher syndrome type 1B xRP (Norrie) Reduction in a- and bwaves Photoreceptor degeneration Gyrate atrophy (chorioretinal degeneration) Prolonged implicit time of b-wave

48 49, 50 51

52 53

54, 55 56, 57

58 59

60

61

62

a Rho, Rhodopsin; RP, retinitis pigmentosa; ad, autosomal dominant; ar, autosomal recessive; x, x linked; CSNB, congenital stationary night blindness; MD, macular degeneration; ND, Norrie disease; prcd, progressive rod/cone degeneration; rd, retinal degeneration; rcd, rod/cone dysplasia; OAT, ornithine aminotransferase; LCA, Leber's Congenital Amaurosis. b Gene and the function of the gene product, if known. c Animal model (naturally occurring or laboratory generated). d Gene defect (amino acid substitutions, or nucleotide deletions), if known. e Key to references: (1) J. E. Olsson, J. W. Gordon, B. S. Pawlyk et aL, Neuron 9, 815 (1992); (2) M. I. Naash, J. G. Hollyfield, M. R. A1-Ubaidi, and W. Baehr, Proc. Natl. Acad. Sci. U.S.A. 90, 5499 (1993); (3) T. Li, M. A. Sandberg, B. S. Pawlyk et aL, Proc, Natl. Acad. Sci. U.S.A. 95, 11933 (1998); (4) T. Li, W. K. Snyder, J. E. Olsson, and T. P. Dryja, Proc. Natl. Acad, Sci. U.S.A. 93, 14176 (1996); (5) P. C. Huang, A. E. Gaitan, Y. Hao, R. M. Petters, and F. Wong, Proc. Natl. Acad. Sci. U.S.A. 90, 8484 (1993); (6) C.-H, Sung, C. Makino, D. Baylor, and J. Nathans, J. Neurosci. 14, 5818 (1994); (7) C. Portera-Cailliau, C.-H. Sung, J. Nathans, and R. Adler, Proc. Natl. Acad. Sci. U.S.A. 91, 974 (1994); (8) P. A. Sieving, J. E. Richards, F. Naarendorp, E. L. Bingham, K. Scott, and (continued)

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Visual Sci. 36, $641 (1995); (29) J. Xu, R. L. Dodd, C. L. Makino, M. I. Simon, D. A. Baylor, and J. Chen, Nature (London) 389, 505 (1997); (30) A. L. Lyubarski, E. N. Pugh, B. Falsini, P. Valentini and J. Chen, Invest. Ophthalmol. Visual Sci. 39. $643 (1998); (31) A. L. Lyubarski, C.-K. Chen, M. I. Simon, and E. N. Pugh, Jr., Invest. OphthalmoL Visual Sci. 40, $390 (1999); (32) G. I. Liou, Y. Fei, N. S. Peachey et aL, J. Neurosci. 18, 4511 (1998); (33) G. D. Aguirre, V. Baldwin, S. Pearce-Kelling, K. NarfstrOm, K. Ray, and G. M. Acland, Mol. Vis. 4, 23 (1998); (34) A. Veske, S. E. Nilsson, K. NarfstrOm, and A. Gal, Genomics, 57, 57 (1999); (35) T. M. Redmond, S. Yu, E. Lee, D. Bok, D. Hamasaki, and K. Pfeifer, Invest. OphthalmoL Visual Sci. 39, $643 (1998); (36) T. M. Redmond, S. Yu, E. Lee et aL, Nature Genet. 20, 344 (1998); (37) J. Weng, S. M. Azarian, and G. H. Travis, Invest. OphthaL Visual Sci. 39, $643 (1998); (38) G. H. Travis, J. G. Sutcliffe, and D. Bok, Neuron 6, 61 (1991); (39) G. H. Travis, M. B. Brennan, P. E. Danielson, C. A. Kozak, and J. G. Sutcliffe, Nature (London) 338, 70 (1989); (40) G. A. Clarke, J. Rossant, and R. R. McInnes, Invest. Ophthalmol. Visual Sci. 39, $962 (1998); (41) R. L. Sidman, B. Kosaras, and M. Tang, Invest. Ophthalmol. Visual Sei. 37, 1097 (1996); (42) B. L. Evans and S. B. Smith, Mol. Vis. 3, 11 (1997); (43) R. J. Mullen and M. M. LaVail, Science 192, 799 (1976); (44) M. J. McLaren and G. Inana, FEBS Lett. 412, 2l (1997); (45) R. Curtis, K. C. Barnett, and A. Leon, Invest. Ophthalmol. Visual Sci. 28, 131 (1987); (46) G. M. Acland, K. Ray, C. S. Mellersh et aL, Proc. Natl. Acad. Sci. U.S.A. 95, 3048 (1998); (47) Q. Zhang, G. M. Acland, C. J. Parshall, J. Haskell, K. Ray, and G. D. Aguirre, Gene 215, 231 (1998); (48) K. Ray, V. J. Baldwin, C. Zeiss, G. M. Acland, and G. D. Aguirre, Curr. Eye Res. 16, 71 (1997); (49) A. Nomura, R. Shigemoto, Y. Nakamura, N. Okamoto, N. Mizuno, and S. Nakanishi, Cell 77, 361 (1994); (50) M. Masu, H. Iwakabe, Y. Tagawa, et aL, Cell 80, 757 (1995); (51) L. Li and J. E. Dowling, Proc. Natl. Acad. Sci. U.S.A. 94,11645 (1997); (52) M. T. Pardue, M. A. McCall, M. M. LaVail, R. G. Gregg, and N. S. Peachey, Invest. Ophthalmol. Visual Sci. 39, 2443 (1998); (53) L. Feng, A. B. Seymour, S. Jiang et al., Hum. MoL Genet. 8, 323 (1999); (54) J. A. van den Hurk, W. Hendriks, D. J. van de Pol et al., Hum. Mol. Genet 6, 851 (1997); (55) J. A. van den Hurk, M. Schwartz, H. van Bokhoven et al., Hum. Mutat. 9, 110 (1997); (56) P. Mburu, X. Z. Liu, J. Walsh et aL, Genes Funct. 1, 191 (1997); (57) F. Gibson, J. Walsh, P. Mburu et al., Nature (London) 374, 62 (1995); (58) W. Berger, D. van de Pol, D. Baehner et al., Hum. Mol. Genet 5, 51 (1996); (59) H. Jiang, A. Lyubarsky, R. Dodd et al., Proc. Natl. Acad. Sci. U.S.A. 93, 14598 (1996); (60) C. Ma, D. Papermaster, and C. L. Cepko, Proc. Natl. Acad. Sci. U.S.A. 95, 9938 (1998); (61) T. Wang, A. M. 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Gene Defects Affecting Levels of cGMP Because a number of animal models (Fig. 1) are associated with mutations in genes affecting cGMP metabolism, we summarize briefly the underlying gene defects and their consequences. The major components are cGMP phosphodiesterase (PDE) and the GC-GC-activating protein (GCAP) complex. Naturally occurring homozygous PDE~ null alleles cause retinal degeneration in the rd mouse, 6 and the rcdl Irish setter. 7 A laboratory-generated PDEr knockout resulted in a phenotype that was similar to that of rd. 8 In these animal models, PDE~ or PDE~ was not produced, and PDE was found to be inactive, leading to elevated levels of cytoplasmic cGMP. A point mutation in PDEr, reintroduced into homozygous PDEr knockouts, impaired transducin-PDE interactions and slowed the recovery rate of the flash response in transgenic mouse rods. 9 A GCI null mutation was linked to the retinal degeneration (rd) chicken 1° (and see [35] in this volumell), a phenotype nearly identical to that of human Leber's congenital amaurosis (LCA), 12 a severe retinopathy of the heterogeneous RP group. In the rd chicken, GC is inactive and cGMP is not produced to levels sufficient to sustain phototransduction. Both the GC1 gene 13and the GCAP genes TM have been knocked out in animal models. Preliminary results indicate that loss of GCI function primarily affects cones, and loss of GCAPI/ GCAP2 affects Ca 2+ sensitivity of recovery. TM Diagnostic Tests (Genotyping) of Naturally Occurring or Transgenic/ Knockout Mouse Models To identify whether a particular mouse strain carries a known mutation (e.g., rd), a rapid diagnostic test is most helpful. The best diagnostic tests available are based on polymerase chain reaction (PCR) amplification of specific exons, and direct sequencing or restriction digests of amplified products. In most cases this procedure requires isolation of genomic DNA (in mice mostly from tail clippings), amplification of gene fragments or exons with specific oligonucleotide primers, digestion of the fragment with 6 S. J. Pittler and W. Baehr, Proc. Natl. Acad. Sci. U.S.A. 88, 8322 (1991). 7 M. L. Suber, S. J. Pittler, N. Qin et. al., Proc. Natl. Acad. Sci. U.S.A. 90, 3968 (1993). 8 S. H. Tsang, P. Gouras, C. K. Yamashita et al., Science 272, 1026 (1996). 9 S. H. Tsang, M. E. Burns, P. D. Calvert et al. Science 282, 117 (1998). 10 S. L. Semple-Rowland, N. R. Lee, J. P. Van-Hooser, K. Palczewski, and W. Baehr, Proc. Natl. Acad. Sci. U.S.A. 95, 1271 (1998). n S. L. Semple-Rowland and N. R. Lee, Methods Enzymol. 316, Chap. 35, 2000 (this volume). 12 I. Perrault, J.-M. Rozet, P. Calvas et al., Nature Genet. 14, 461 (1996). 13 D. G. Birch, R.-B. Yang, and D. L. Garbers, Invest. Ophthalmol. Vis. Sci. 39, $643 (1998). 14 A. Mendez, M. E. Burns, I. Sokal et al., Invest. Ophthalmol. Vis. Sci. 40 (1999).

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a diagnostic restriction enzyme, and/or direct sequencing or sequencing after cloning. The following is a set of methods serving these purposes. Isolation of Genomic DNA from Mouse Tail Biopsies

Required Materials Clean, sterile scalpel blades Microcentrifuge tubes, 1.5 ml Tail buffer [50 mM Tris (pH 7.5), 50 mM E D T A (pH 8.0), 100 mM NaC1, 5 mM dithiothreitol (DTT), 0.5 mM spermidine, sodium dodecyl sulfate (SDS; 2%, w/v)] Proteinase K, 20 mg/ml in water Potassium acetate, 3 M/5 M 2-Propanol Ethanol (70%, v/v) Tris-EDTA (TE, 1 ×) Water bath, 55 ° To prepare 100 ml of tail buffer, combine Tris (pH 7.5), 1.0 M 5 ml E D T A (pH 8.0), 0.5 M 10 ml NaCI, 5.0 M 2 ml DT-I', 1.0 M 0.5 ml Spermidine, 1.0 M 50 ~1 SDS 2g Bring the volume to 100 ml with sterile, deionized water.

Procedure Using a scalpel blade dipped in ethanol and flamed, cut 1-2 cm of distal tail and place it in a microcentrifuge tube. Add 0.6 ml of tail buffer and 20/xl of proteinase K to each tube. Incubate overnight at 55° with gentle shaking. Vortex each sample briefly to ensure that tails are digested. Pellet cellular debris by spinning the tubes at 15,000 rpm for 2 min at 4 °. Transfer each supernatant to a clean 1.5-ml microcentrifuge tube, add 200/LI of potassium acetate solution, and vortex vigorously for 20 sec. Chill the samples on ice for 5 min to precipitate the proteins. Centrifuge for 4 min at 15,000 rpm at 4 °. Decant the supernatant into a clean 1.5-ml microcentrifuge tube containing 0.6 ml of 2-propanol. Precipitate the D N A by inverting each tube until a white mass forms. Centrifuge at 15,000 rpm for 1 min at 4°. Pour off the supernatant, add 0.6 ml of 70% (v/v) ethanol, and wash

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the D N A pellet by inverting the tube several times. Centrifuge at 15,000 rpm for 1 min at 4 °. Carefully decant the ethanol and air dry the D N A pellet for 15-20 min. Add 100/xl of TE and dissolve the pellet overnight at room temperature. The yield of nucleic acid approaches 1 ~g//xl as estimated by agarose gel electrophoresis. Refrigerate at 4 ° for near-term analysis, or freeze at - 2 0 ° for long-term storage. Although this DNA preparation is suitable for PCR amplification on appropriate dilution (1 : 25), trace amounts of salt and detergent may require reextraction with phenol-chloroform if, for example, the D N A is to be used for Southern blots and/or restriction digests.

Genomic Polymerase Chain Reaction for Genotyping An excellent, practical overview of genotyping requirements and techniques used in support of transgenic colonies is availableJ 5 A stringent protocol for amplifications of genomic fragments takes into consideration the melting points of the oligonucleotide primers used, the length of the fragment to be amplified, other parameters such as MgCI2 and pH sensitivity of given primer pairs, and peculiarities of the PCR cycler. The following are two sample protocols used in our laboratory for amplification of genomic DNA.

Example 1: Amplification of Diagnostic Exon 7 of Mouse PDE~ Subunit Gene A nonsense ochre mutation (T ~ A transversion in codon 347 of the PDEe gene) is responsible for the rd mouse phenotype. 6 The nonsense mutation introduces a new DdeI site that can be used in subsequent enzymatic digestions to verify its presence or absence. The pimer pair amplifies a 296-bp fragment that carries two DdeI sites in the rd PDE~ subunit allele and none in the normal allele. The amplification requires 5-20 pmol each of primer 1 (sense, 5'-CAT CCC ACC T G A GCT CAC A G A AAG) and primer 2 (antisense, 5'-GCC TAC A A C A G A G G A GCT TCT AGC), 25-75 ng of genomic DNA, and standard buffer conditions. Cycling is done 35 times at 92° for 30 sec, 55° for 30 sec, and 72 ° for 30 sec in a Perkin-Elmer, (Norwalk, CT) GeneAmp 960 cycler (or equivalent). A single fragment of the indicated size can be detected in agarose gels. The fragment is subsequently digested with DdeI as described below.

15 B. Elder, Lab. Anim. 20 (1999).

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Example 2: Amplification of Exon 1 of Mouse Rhodopsin Gene for Genotyping A transgenic mouse line with three mutations in exon 1 and a restriction fragment length polymorphism (RFLP) deleting an intrinsic NcoI site was generated. I6 The diagnostic assay consists of amplification of exon 1 of the mouse rhodopsin gene, followed by NcoI digestion. Amplification with primers W75/Wll produces a 1.3-kb fragment, which must first be verified by electrophoresis on a 1% (w/v) agarose gel. After digestion of the fragment with NcoI (typical reaction given below), wild-type mice reveal three fragments of 689, 431, and 197 bp. Transgenic (heterozygous) mice have, in addition, an 886-bp fragment (689 + 197 bp) owing to the deletion of one NcoI site in the transgene.

Restriction Digest of Amplified Fragments Restriction enzymes are supplied with 10x buffers for which activity is optimal. The conditions (pH, salt) optimal for Taq polymerase activity may, or may not, be compatible with those of the restriction enzyme. To minimize potential incompatibility, an aliquot of the amplification reaction is diluted in a restriction digest of larger volume (20/zl), e.g., to 12/zl of distilled H20, one adds 2/.d of 10x buffer, 1/zl of enzyme and 5/.d of amplification reaction, mixing after each addition. After incubation according to the enzyme manufacturer recommendation (e.g., 1 hr at 37°), the products of digestion are separated electrophorectically by loading 10/zl of the restriction digest per lane of a 1-2% (w/v) agarose gel. Small fragment sizes (<500 bp) are resolved more readily in gels of 2% (w/v) agarose concentration.

Sequencing of Amplified Fragments If diagnostic restriction sites are not present, the DNA fragment must be sequenced directly to assess for mutations. The most common procedures sequence a DNA fragment directly after amplification, or after cloning. Most sequencing today is done with automatic sequencers and chemistry based on linear PCR (one primer only), primer extension, and chain terminators.

Direct Sequencing By far the most used method is based on the Perkin-Elmer/Applied Biosystems large-scale sequencers available through core facilities. On a 16 M. I. Naash, J. G. Hollyfield, M. R. A1-Ubaidi, and W. Baehr, Proc. Natl. Acad. ScL U.S.A. 90, 5499 (1993).

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smaller scale, we found the Perkin-Elmer ABI 310 capillary sequencer to be the most reliable and useful. The ABI 310 works without gels, separates DNA fragments by capillary electrophoresis, and provides sequences up to 400-bp fragments in less than 2 hr. For all sequencing, the DNA template should be highly purified, using for example, a Qiagen (Chatsworth, CA) DNA preparation kit or a cesium chloride gradient. ABI 310 Protocol: Two hundred to 500 ng of plasmid DNA template (or 30-90 ng of amplified fragment) is needed for each reaction. The primer extension chemistry uses labeled dideoxy terminators, each labeled with a different fluorescent dye emitting light at distinct wavelengths. Consequently, each reaction can be done in a single tube. Unincorporated terminators, however, will create a background during the electrophoresis, and must be removed by precipitation or column chromatography before loading the sample onto the capillary. Perkin-Elmer/Applied Biosystems markets a kit for this chemistry called the BigDye Terminator Cycle Sequencing Ready Reaction kit. To set up a typical reaction, combine H20 15.0/xl DNA template 1.0/zl Primer (3.2 pmol) 1.0/.d Ready Reaction Mix 8.0/xl 20.0/zl Procedure. A layer of oil is not needed over the reactions if a thermal cycler with a heated top is used. A basic cycling program for a PerkinElmer 9700 thermal cycler consists of a denaturing step (94° for 2 min), and 25 cycles of 94 ° for 10 sec, 50 ° for 5 sec, and 60 ° for 4 min. After cycling is complete, the reaction is passed through a column [Centri-Sep column (Princeton Separations, Adelphia, NJ) or Sephadex G-50 column] to remove excess unincorporated nucleotides. Desiccate the flow-through in a SpeedVac concentrator and add 25/xl of template suppression reagent (supplied with the kit); then load the sample into the sequencer for electrophoresis through the capillary.

Plasmid Sequencing after Cloning of Genomic Fragments For large fragments (>3 kb), cloning followed by sequencing most often yields the best results. The fragment of interest can be cloned into PCR vectors such as PCR2.1 (InVitrogen, San Diego, CA), and the resulting plasmid sequenced with universal primers and automatic sequencers. The following example describes a cycle-sequencing method using a Li-Cor DNA4000 automatic sequencer (Li-Cor Technologies, Lincoln, NE). This system uses infrared-tagged primers, requires four lanes per reaction, and

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employs long polyacrylamide-based gels for separation of the DNA fragments. The Li-Cor sequences reliably more than 1 kb in one direction once parameters have been optimized. Li-Cor DNA4000 Protocol Two hundred to 1000 ng of template is needed for each reaction (depending on plasmid size). Relatively inexpensive prelabeled standard primers as well as custom labeled primers may be obtained directly from Li-Cor Technologies. Because the primer is prelabeled in this chemistry, termination reactions must be done (and loaded onto the gel) separately. Epicentre Technologies (Madison, WI) markets a kit for this chemistry called SequiTherm Excell II. A typical reaction includes H20 7.8/.d DNA template (0.2 pmol) 1.0/zl Labeled Primer (2 pmol) 1.0/.d Excel II reaction buffer 7.2/zl SequiTherm Excel II enzyme 1.0/zl 18.0/.d G, A, T, and C termination reactions are prepared by adding 2/zl of termination mix (supplied with the kit) to PCR tubes labeled accordingly. Transfer 4-/xl aliquots into the four termination reaction tubes. A basic linear cycling program for a Perkin-Elmer 9700 thermal cycler consists of the following steps: (1) denature--94 ° for 5 min; (2) cycle (25 cycles)--94 ° for 30 sec, 50° for 15 sec, and 70° for 1 min. After cycling is complete, add 4/zl of loading buffer (supplied with kit) to each termination reaction, and load 1.5 /zl of each onto the gel to electrophorese. Six hundred to 1200 bases of sequence can be obtained per reaction. Data acquisition is fully automatic.

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Avian Models of Inherited Retinal Disease

By S U S A N

L. SEMPLE-ROWLAND

and NANCY R. LEE

Introduction There has been a dramatic increase in the number of genes that have been linked to inherited retinal disease (see RetNet at http://www.sph.uth. tmc.edu/Retent), many of which encode proteins that directly affect the function of photoreceptor cells. The most devastating human retinal dystrophies affect cone cells, the photoreceptor cells responsible for color perception and high visual acuity in humans. Avian species, and in particular the

METHODS IN ENZYMOLOGY, VOL. 316

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