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J. R. Sommer, V. R. Chavali, R. M. Petters, R. Ayyagari; Cloning and Expression Analysis of Pig CTRP5 (Complement-1q Tumor Necrosis Factor- Related Protein 5) Gene. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2349.
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© ARVO (1962-2015); The Authors (2016-present)
Autosomal dominant early-onset long anterior zonules (LAZ) and late-onset retinal degeneration (L-ORD) are associated with the S163R mutation of the CTRP5 gene. For the purpose of utilizing the pig as a transgenic model for the study of CTRP5 pathology, we cloned, characterized, developed transgenic vectors and studied the endogenous expression profile of pig CTRP5.
The pig CTRP5 gene (pCTRP5) was cloned and sequenced from porcine genomic DNA. V5 epitope tagged constructs of the pCTRP5 genes as well as the mammalian promoters: elongation factor 1- (EF) promoter, mouse retinal pigment epithelium 65 (RPE65) and 579 bp of the putative promoter located upstream to pCTRP5 DNA were used for in vitro expression analysis. pCTRP5 transgene expression, protein size and cellular localization were assayed using immunocytochemistry (ICC) and western blot analysis on transiently transfected CHO-K1, Cos7 or ARPE-19 cells using CTRP5 and V5 specific antibodies. Expression of endogenous pCTRP5 in the pig eye was analyzed by western blot analysis and real time PCR. For the purpose of making a transgene, the S163R mutation was made in the pCTRP5 gene using PCR mediated site-directed mutagenesis.
The pCTRP5 gene shows a 92% DNA homology and 98% amino acid homology with human CTRP5. The putative endogenous pCTRP5 promoter was found to be functional. The level of expression of V5 tagged pCTRP5 was found to be similar under the regulation of the three (EF promoter, RPE65 promoter and 579 bp of pCTRP5) promoters tested. Pig CTRP5 showed cytoplasmic localization in ARPE19 cells that are transiently transfected with V5 pCTRP5 constructs. The size of the pig CTRP5 protein is consistent with its predicted molecular weight. Western blot and RT/PCR analysis shows that pCTRP5 is predominantly expressed in RPE, a pattern of expression consistent with that found in mouse and human eyes.
The sequence, genomic organization and protein sequence of pig CTRP5 gene is similar to its human homolog. We identified a putative promoter region upstream to the pCTRP5 which is capable of controlling its expression. The tissue distribution and expression profile of pig CTRP5 is consistent with human CTRP5. The expression of pCTRP5 was found to be similar when cloned downstream to all three promoters. Due to these similarities the domestic pig will make a good model for study of LAZ and L-ORD.
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