To clone the first anion channel from Xenopus laevis (X. laevis), we isolated a calcium-activated chloride channel (CLCA)-like membrane protein 6 gene (CMP6) in X. laevis. As a first step in gene isolation, an expressed sequence tags database was screened to find the partial cDNA fragment. A putative partial cDNA sequence was obtained by comparison with rat CLCAs identified in our laboratory. First stranded cDNA was synthesized by reverse transcription polymerase-chain reaction (RT-PCR) using a specific primer designed for the target cDNA. Repeating the 5' and 3' rapid amplification of cDNA ends, full-length cDNA was constructed from the cDNA pool. The full-length CMP6 cDNA completed via 5'- and 3'-RACE was 2,940 bp long and had an open reading frame (ORF) of 940 amino acids. The predicted 940 polypeptides have four major transmembrane domains and showed about 50% identity with that of rat brain CLCAs in our previously published data. Semi-quantification analysis revealed that CMP6 was most abundantly expressed in small intestine, colon and liver. However, all tissues except small intestine, colon and liver had undetectable levels. This result became more credible after we did real-time PCR quantification for the target gene. In view of all CLCA studies focused on human or murine channels, this finding suggests a hypothetical protein as an ion channel, an X. laevis CLCA.
Nonhuman primates are important animal models in transplantation. To prevent fatal transplantation-induced immune responses, it is necessary to accurately phenotype the monkey ABH antigens, which are the same as those in humans but (unlike in humans) are not expressed on red blood cells (RBCs). We compared the ability of two established ABO-typing methods, namely, serological testing and immunohistochemistry (IHC), and our novel polymerase chain reaction (PCR)-based assay to type 66 rhesus monkeys. The serological test assessed the ability of monkey sera to hemagglutinate human RBCs. The IHC assay measured the binding of murine anti-A and anti-B antibodies to monkey buccal mucosa cells. The whole blood-based PCR assay involved selective primers that were derived from the exon 7 sequences of A+, B+, and O+ monkeys. IHC and PCR unequivocally yielded the same types in all monkeys. Serological testing yielded inconsistent types in seven (10.6%). FACS analysis with monkey sera preabsorbed with O+ RBCs showed that the incorrect serological results related to nonspecific or xenoreactive binding of the human RBCs. Unlike previous PCR-based assay, our algorithm directly detected O+ monkeys and A and B homozygotes and heterozygotes. Given the logistical limitations of IHC, this PCR assay may be useful for typing rhesus monkeys.
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