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Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank.

Simpanya MF, Wistow G, Gao J, David LL, Giblin FJ, Mitton KP - Mol. Vis. (2008)

Bottom Line: Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank.For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin.In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI 48309, USA.

ABSTRACT

Purpose: To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags.

Methods: RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS).

Results: Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including alphaA/alphaAinsert-, gammaN-, and gammaS-crystallins, lengsin and GRIFIN. The ratio of alphaA- to alphaB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of gammaD-, gammaE-, and gammaF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea.

Conclusions: Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of gammaD-, gammaE-, and gammaF-crystallin were found in EST, proteomic, or the current guinea pig genome data.

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Morphology of guinea pig eye tissues for the Hartley strain used for the NEIBank library, stained with hematoxylin and eosin reagent. A: Lens capsule, epithelium and cortex in the bow region: capsule (CAP), epithelium (EPI), and cortical fiber cells (COR). The guinea pig lens is similar to human and mouse. B: Neural retina (NR), retinal pigment epithelium (RPE), choroid (CHR) and sclera (SCL). C: Retinal layer and choroid: ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS), outer segment (OS), retinal pigment epithelium (RPE), and choroid (CHR). The guinea pig retina is 4–5 nuclei thick, similar to the human retina. D: Cornea: corneal epithelium (EP), Bowman’s membrane (BOW), Stroma (STR), Descemet’s membrane (DES) and endothelium (END). The guinea pig cornea is similar to human, while the mouse has a thinner stroma.
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f1: Morphology of guinea pig eye tissues for the Hartley strain used for the NEIBank library, stained with hematoxylin and eosin reagent. A: Lens capsule, epithelium and cortex in the bow region: capsule (CAP), epithelium (EPI), and cortical fiber cells (COR). The guinea pig lens is similar to human and mouse. B: Neural retina (NR), retinal pigment epithelium (RPE), choroid (CHR) and sclera (SCL). C: Retinal layer and choroid: ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS), outer segment (OS), retinal pigment epithelium (RPE), and choroid (CHR). The guinea pig retina is 4–5 nuclei thick, similar to the human retina. D: Cornea: corneal epithelium (EP), Bowman’s membrane (BOW), Stroma (STR), Descemet’s membrane (DES) and endothelium (END). The guinea pig cornea is similar to human, while the mouse has a thinner stroma.

Mentions: Intact 2.5-month-old guinea pig lenses were used to make the lens cDNA library, designated “nbb.” The guinea pig lens is very similar to the human lens, with a monolayer epithelium, but the guinea pig capsule is thinner than the human capsule . Guinea pig lens capsule, epithelium, and cortex are shown in Figure 1A. Neural retina (Figure 1B,C) was used for the retina cDNA library, designated as “naz.” The guinea pig outer nuclear layer (ONL) is about 5 nuclei thick (photoreceptors), which is more similar to the human ONL (6 nuclei) than the mouse ONL (10–12 nuclei) [51,52]. The eye minus lens and retina cDNA library, NEIBank designation “nba,” included several eye tissues such as choroid (Figure 1B,C), sclera (Figure 1B), RPE (Figure 1B,C), iris, and cornea (Figure 1D).


Expressed sequence tag analysis of guinea pig (Cavia porcellus) eye tissues for NEIBank.

Simpanya MF, Wistow G, Gao J, David LL, Giblin FJ, Mitton KP - Mol. Vis. (2008)

Morphology of guinea pig eye tissues for the Hartley strain used for the NEIBank library, stained with hematoxylin and eosin reagent. A: Lens capsule, epithelium and cortex in the bow region: capsule (CAP), epithelium (EPI), and cortical fiber cells (COR). The guinea pig lens is similar to human and mouse. B: Neural retina (NR), retinal pigment epithelium (RPE), choroid (CHR) and sclera (SCL). C: Retinal layer and choroid: ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS), outer segment (OS), retinal pigment epithelium (RPE), and choroid (CHR). The guinea pig retina is 4–5 nuclei thick, similar to the human retina. D: Cornea: corneal epithelium (EP), Bowman’s membrane (BOW), Stroma (STR), Descemet’s membrane (DES) and endothelium (END). The guinea pig cornea is similar to human, while the mouse has a thinner stroma.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2605723&req=5

f1: Morphology of guinea pig eye tissues for the Hartley strain used for the NEIBank library, stained with hematoxylin and eosin reagent. A: Lens capsule, epithelium and cortex in the bow region: capsule (CAP), epithelium (EPI), and cortical fiber cells (COR). The guinea pig lens is similar to human and mouse. B: Neural retina (NR), retinal pigment epithelium (RPE), choroid (CHR) and sclera (SCL). C: Retinal layer and choroid: ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS), outer segment (OS), retinal pigment epithelium (RPE), and choroid (CHR). The guinea pig retina is 4–5 nuclei thick, similar to the human retina. D: Cornea: corneal epithelium (EP), Bowman’s membrane (BOW), Stroma (STR), Descemet’s membrane (DES) and endothelium (END). The guinea pig cornea is similar to human, while the mouse has a thinner stroma.
Mentions: Intact 2.5-month-old guinea pig lenses were used to make the lens cDNA library, designated “nbb.” The guinea pig lens is very similar to the human lens, with a monolayer epithelium, but the guinea pig capsule is thinner than the human capsule . Guinea pig lens capsule, epithelium, and cortex are shown in Figure 1A. Neural retina (Figure 1B,C) was used for the retina cDNA library, designated as “naz.” The guinea pig outer nuclear layer (ONL) is about 5 nuclei thick (photoreceptors), which is more similar to the human ONL (6 nuclei) than the mouse ONL (10–12 nuclei) [51,52]. The eye minus lens and retina cDNA library, NEIBank designation “nba,” included several eye tissues such as choroid (Figure 1B,C), sclera (Figure 1B), RPE (Figure 1B,C), iris, and cornea (Figure 1D).

Bottom Line: Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank.For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin.In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea.

View Article: PubMed Central - PubMed

Affiliation: Eye Research Institute, Oakland University, Rochester, MI 48309, USA.

ABSTRACT

Purpose: To characterize gene expression patterns in guinea pig ocular tissues and identify orthologs of human genes from NEIBank expressed sequence tags.

Methods: RNA was extracted from dissected eye tissues of 2.5-month-old guinea pigs to make three unamplified and unnormalized cDNA libraries in the pCMVSport-6 vector for the lens, retina, and eye minus lens and retina. Over 4,000 clones were sequenced from each library and were analyzed using GRIST for clustering and gene identification. Lens crystallin EST data were validated using two-dimensional electrophoresis (2-DE), matrix assisted laser desorption (MALDI), and electrospray ionization mass spectrometry (ESIMS).

Results: Combined data from the three libraries generated a total of 6,694 distinctive gene clusters, with each library having between 1,000 and 3,000 clusters. Approximately 60% of the total gene clusters were novel cDNA sequences and had significant homologies to other mammalian sequences in GenBank. Complete cDNA sequences were obtained for many guinea pig lens proteins, including alphaA/alphaAinsert-, gammaN-, and gammaS-crystallins, lengsin and GRIFIN. The ratio of alphaA- to alphaB-crystallin on 2-DE gels was 8: 1 in the lens nucleus and 6.5: 1 in the cortex. Analysis of ESTs, genome sequence, and proteins (by MALDI), did not reveal any evidence for the presence of gammaD-, gammaE-, and gammaF-crystallin in the guinea pig. Predicted masses of many guinea pig lens crystallins were confirmed by ESIMS analysis. For the retina, orthologs of human phototransduction genes were found, such as Rhodopsin, S-antigen (Sag, Arrestin), and Transducin. The guinea-pig ortholog of NRL, a key rod photoreceptor-specific transcription factor, was also represented in EST data. In the 'rest-of-eye' library, the most abundant transcripts included decorin and keratin 12, representative of the cornea.

Conclusions: Genomic analysis of guinea pig eye tissues provides sequence-verified clones for future studies. Guinea pig orthologs of many human eye specific genes were identified. Guinea pig gene structures were similar to their human and rodent gene counterparts. Surprisingly, no orthologs of gammaD-, gammaE-, and gammaF-crystallin were found in EST, proteomic, or the current guinea pig genome data.

Show MeSH
Related in: MedlinePlus