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Polyclonal antibody was purified from serum with thiophilic adsorbent resin according to manufacturer’s instructions (Thermo Scientific, Waltham, MA)

Polyclonal antibody was purified from serum with thiophilic adsorbent resin according to manufacturer’s instructions (Thermo Scientific, Waltham, MA). by the absorption of photons by visual pigments within the eye’s retina. Consisting of the 11-mice. Results “type”:”entrez-nucleotide”,”attrs”:”text”:”BC027072″,”term_id”:”20072553″,”term_text”:”BC027072″BC027072 expression analysis Data mining as well as previous studies suggest that “type”:”entrez-nucleotide”,”attrs”:”text”:”BC027072″,”term_id”:”20072553″,”term_text”:”BC027072″BC027072 is almost exclusively expressed in the eye, with a significantly lower expression found in the testes, which are known to have a relaxed chromatin structure leading to aberrant expression (25). Previous studies relied on semi-quantitative polymerase chain reaction (qRT-PCR) or microarray analysis for gene expression analyses. To provide a more thorough analysis, we used qRT-PCR to analyze gene expression in a range of mouse tissues as well as in different compartments of the eye. Our analysis confirms previous reports with the eye manifesting significantly higher expression than any other tissue (Fig.?2A) (18), with a low amount of expression found in the testes, consistent with other studies. Within the eye, expression was confined to the neural retina, with little or no expression detected in the RPE/choroid tissues (Fig.?2B). CHMFL-ABL-121 To understand the expression of this gene in the mouse eye, we mined our recently analyzed RNASeq transcriptome data from various species and tissues. In rod dominant rodents, “type”:”entrez-nucleotide”,”attrs”:”text”:”BC027072″,”term_id”:”20072553″,”term_text”:”BC027072″BC027072 was expressed at levels comparable to those of ABCA4, an important component of the retinoid cycle in the retina (26) (Table?1). Comparison of expression levels across species, including those with varying numbers of rods and cones, suggests AKT2 that this gene is expressed in both rod and cone photoreceptor cells. Table?1. Gene expression analysis by RNAseq displayed a clear single band. Analysis of native eye tissue from mice with our pAb yielded a distinct band of the appropriate size, which was seen only CHMFL-ABL-121 in samples that included retinal tissue (Fig.?3A). Analysis of cryosections by immunohistochemistry (IHC) revealed a photoreceptor-specific localization, with staining in the IS of both rod and cone photoreceptors and a significant level of expression in the OS of what appeared to be cone photoreceptors. Co-labeling with peanut agglutinin (PNA) or cone opsin confirmed the CHMFL-ABL-121 expression in cone OS (Fig.?3B). qRT-PCR analysis of RNA from the coneless transgenic line (27), ConeDTA, which expresses diphtheria toxin A under CHMFL-ABL-121 a cone-specific promoter, showed robust expression of “type”:”entrez-nucleotide”,”attrs”:”text”:”BC027072″,”term_id”:”20072553″,”term_text”:”BC027072″BC027072, ruling out a cone-specific expression. Open in a separate window Figure?3. Immunoblot and IHC localization of “type”:”entrez-nucleotide”,”attrs”:”text”:”BC027072″,”term_id”:”20072553″,”term_text”:”BC027072″BC027072 in mouse eye with a custom pAb. (A) Tissues were dissected and proteins were extracted with RIPA buffer. Immunoblot analysis confirmed the qRT-PCR analysis and demonstrated that expression is confined to the neural retina within the eye. rBC027072 is a truncated recombinant protein (amino acids 1C360) expressed in which was not used for immunization. (B) Eyes from 3-week-old WT and mice were fixed in paraformaldehyde and embedded in OCT compound. Cryosections (12 m) were probed with a “type”:”entrez-nucleotide”,”attrs”:”text”:”BC027072″,”term_id”:”20072553″,”term_text”:”BC027072″BC027072 pAb and PNA (for cone cell staining). Staining of “type”:”entrez-nucleotide”,”attrs”:”text”:”BC027072″,”term_id”:”20072553″,”term_text”:”BC027072″BC027072 is consistent with localization to the IS of both cone and rod photoreceptors as well as the OS of cone cells. Early-onset retinal degeneration in mice Next, we generated a knockout mouse line by replacing exon 1 ( 95% of the coding region of “type”:”entrez-nucleotide”,”attrs”:”text”:”BC027072″,”term_id”:”20072553″,”term_text”:”BC027072″BC027072) with a neomycin cassette (Fig.?4A and B). Crossing mice provided the CHMFL-ABL-121 expected Mendelian ratios with no apparent effect on viability. Additionally, viability appeared normal in mating pairs of two homozygous mice with litter sizes comparable to those seen for mating pairs of heterozygous mice, consistent with the tissue specificity of this gene. Analysis of gene expression in the eye by qRT-PCR showed a virtually complete lack of transcripts in mice (Fig.?4C). In mice, outer nuclear layer (ONL) thinning could be seen as early as 4 weeks of age. As analyzed by spectral domain optical coherence tomography (SD-OCT), ONL thickness had already decreased 25% by 4 weeks of age and rapidly degenerated further with 42 and 81% of the retina lost by 8 and 24 weeks of age, respectively (Fig.?5A and C). Histological examination of plastic-embedded tissues not.