Dataset Information

Identification of novel MHC class I sequences in pig-tailed macaques by amplicon pyrosequencing and full-length cDNA cloning and sequencing.

ABSTRACT: Pig-tailed macaques (Macaca nemestrina) provide important animal models in biomedical research, but utility of this species for HIV and other disease pathogenesis research is limited by incomplete knowledge of major histocompatibility complex (MHC) class I genetics. Here, we describe comprehensive MHC class I genotyping of 24 pig-tailed macaques, using pyrosequencing to evaluate a 367- bp complementary DNA (cDNA)-PCR amplicon spanning the highly polymorphic peptide-binding region of MHC class I transcripts. We detected 29 previously described Mane transcripts, 90 novel class I sequences, and eight shared MHC class IB haplotypes. We used this genotyping data to inform full-length MHC class I cDNA allele discovery, characterizing 66 novel full-length transcripts. These new full-length sequences nearly triple the number of Mane-B cDNA sequences previously characterized. The comprehensive genotypes and full-length Mane transcripts described herein add value to pig-tailed macaques as model organisms in biomedical research; furthermore, the coordinated method for MHC genotyping and allele discovery is extensible to other less well-characterized nonhuman primate species.

SUBMITTER: O'Leary CE

PROVIDER: S-EPMC2813950 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of novel MHC class I sequences in pig-tailed macaques by amplicon pyrosequencing and full-length cDNA cloning and sequencing.

O'Leary Claire E CE Wiseman Roger W RW Karl Julie A JA Bimber Benjamin N BN Lank Simon M SM Tuscher Jennifer J JJ O'Connor David H DH

Immunogenetics 20090924 10

Pig-tailed macaques (Macaca nemestrina) provide important animal models in biomedical research, but utility of this species for HIV and other disease pathogenesis research is limited by incomplete knowledge of major histocompatibility complex (MHC) class I genetics. Here, we describe comprehensive MHC class I genotyping of 24 pig-tailed macaques, using pyrosequencing to evaluate a 367- bp complementary DNA (cDNA)-PCR amplicon spanning the highly polymorphic peptide-binding region of MHC class I ...[more]

PMID: 19777225

Similar Datasets

Project description:Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not 'finished'. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.

Dataset Information

Identification of novel MHC class I sequences in pig-tailed macaques by amplicon pyrosequencing and full-length cDNA cloning and sequencing.

Publications

Identification of novel MHC class I sequences in pig-tailed macaques by amplicon pyrosequencing and full-length cDNA cloning and sequencing.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets