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Expression sequence tag library derived from peripheral blood mononuclear cells of the chlorocebus sabaeus.

Tchitchek N, Jacquelin B, Wincker P, Dossat C, Silva CD, Weissenbach J, Blancher A, Müller-Trutwin M, Benecke A - BMC Genomics (2012)

Bottom Line: For 506 transcripts, sequences were quasi-complete.In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes.Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut des Hautes Études Scientifiques-Centre National de la Recherche Scientifique, Bures-sur-Yvette, France.

ABSTRACT

Background: African Green Monkeys (AGM) are amongst the most frequently used nonhuman primate models in clinical and biomedical research, nevertheless only few genomic resources exist for this species. Such information would be essential for the development of dedicated new generation technologies in fundamental and pre-clinical research using this model, and would deliver new insights into primate evolution.

Results: We have exhaustively sequenced an Expression Sequence Tag (EST) library made from a pool of Peripheral Blood Mononuclear Cells from sixteen Chlorocebus sabaeus monkeys. Twelve of them were infected with the Simian Immunodeficiency Virus. The mononuclear cells were or not stimulated in vitro with Concanavalin A, with lipopolysacharrides, or through mixed lymphocyte reaction in order to generate a representative and broad library of expressed sequences in immune cells. We report here 37,787 sequences, which were assembled into 14,410 contigs representing an estimated 12% of the C. sabaeus transcriptome. Using data from primate genome databases, 9,029 assembled sequences from C. sabaeus could be annotated. Sequences have been systematically aligned with ten cDNA references of primate species including Homo sapiens, Pan troglodytes, and Macaca mulatta to identify ortholog transcripts. For 506 transcripts, sequences were quasi-complete. In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes.

Conclusions: The EST library we provide here will prove useful in gene annotation efforts for future sequencing of the African Green Monkey genomes. Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

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Representation of the “Interferon Signaling” and “Toll-like Receptor Signaling” pathways. (A) Representation of the “Interferon Signaling” pathway. (B) Representation of the “Toll-like Receptor Signaling” pathway. Genes present in the EST library are shown in gray.
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Figure 5: Representation of the “Interferon Signaling” and “Toll-like Receptor Signaling” pathways. (A) Representation of the “Interferon Signaling” pathway. (B) Representation of the “Toll-like Receptor Signaling” pathway. Genes present in the EST library are shown in gray.

Mentions: In order to have a quantitative view of the expressed sequences of the C. sabaeus PBMC, we identified the most expressed transcripts in our EST library based on the M. mulatta homolog transcripts. Based on the 44,725 transcripts of the M. mulatta cDNA reference and the 14,410 ESTs of the original ESTs library, we calculated for each transcript the number of sequences mapped and obtained a list of the 50 most expressed M. mulatta ortholog transcripts in our EST library (Table 3). Among these most expressed transcripts, the hemoglobin beta (HBB) and alpha (HBA) genes were present, which might reflect a red blood cells contaminations of the PBMC, as well as more specific immune-related genes, such as CD74 and Granzyme B (GZMB). Some EST which correspond to genes which play an important role in immune responses against pathogens have been aligned: IRF7 (Figure 4), CD4 (Additional file 4: Figure S3), IFNG (Additional file 5: Figure S4), IFNGR1 (Additional file 6: Figure S5), IFNGR2 (Additional file 7: Figure S6). For all these transcripts, EST alignment positions as well as protein domains are given. Furthermore, in order to identify the over-represented pathways in our AGM EST library, we performed a functional canonical pathway analysis based on the list of the 9,208 H. sapiens transcripts uniquely mapped by the 37,787 original ESTs. Most of the canonical pathways found as statistically significantly over-represented are related to B and T cell signaling, and immune response pathways (Table 4). For instance, the “CD28 signaling in T Helper Cells”, “iCOS-iCOSL signaling in T Helper Cells”, “B Cell receptor Signaling” (Additional file 8: Figure S7A), and “T Cell receptor signaling” (Additional file 8: Figure S7B) pathways belong to the list of pathways found as significantly over-represented in our AGM library, as well as the “Glucocorticoid receptor signaling”, “Role of NFAT in regulation of the immune response” (Additional file 9: Figure S8A), “Antigen presentation pathway” (Additional file 9: Figure S8B), “JAK/STAT signaling”, and many different “Interleukin signaling” pathways. As a result of the in vitro stimulation of SIV-infected PBMC, the “NF-κB Activation by viruses” (Additional file 10: Figure S9A) and “Induction of apoptosis by HIV-1” (Additional file 10: Figure S9B) pathways are also significantly over-represented. Consistent with the stimulation by LPS, the “Interferon Signaling” (Figure 5A) and “Toll-like Receptor Signaling” (Figure 5B) pathways are also found significantly over-represented. Finally, ConA is capable of triggering positive selection in mature T cells by cross-linking the TCR with high avidity [40,41] and we found 8 pathways corresponding to these functions being induced (Table 4). The over-representation of gene transcripts belonging to these pathways of the immune system further indicates that this library is a valuable resource for profiling global gene expression in AGM immune cells. Overall, these gene and pathway information are consistent with what we could expect from an EST PBMC library.


Expression sequence tag library derived from peripheral blood mononuclear cells of the chlorocebus sabaeus.

Tchitchek N, Jacquelin B, Wincker P, Dossat C, Silva CD, Weissenbach J, Blancher A, Müller-Trutwin M, Benecke A - BMC Genomics (2012)

Representation of the “Interferon Signaling” and “Toll-like Receptor Signaling” pathways. (A) Representation of the “Interferon Signaling” pathway. (B) Representation of the “Toll-like Receptor Signaling” pathway. Genes present in the EST library are shown in gray.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539953&req=5

Figure 5: Representation of the “Interferon Signaling” and “Toll-like Receptor Signaling” pathways. (A) Representation of the “Interferon Signaling” pathway. (B) Representation of the “Toll-like Receptor Signaling” pathway. Genes present in the EST library are shown in gray.
Mentions: In order to have a quantitative view of the expressed sequences of the C. sabaeus PBMC, we identified the most expressed transcripts in our EST library based on the M. mulatta homolog transcripts. Based on the 44,725 transcripts of the M. mulatta cDNA reference and the 14,410 ESTs of the original ESTs library, we calculated for each transcript the number of sequences mapped and obtained a list of the 50 most expressed M. mulatta ortholog transcripts in our EST library (Table 3). Among these most expressed transcripts, the hemoglobin beta (HBB) and alpha (HBA) genes were present, which might reflect a red blood cells contaminations of the PBMC, as well as more specific immune-related genes, such as CD74 and Granzyme B (GZMB). Some EST which correspond to genes which play an important role in immune responses against pathogens have been aligned: IRF7 (Figure 4), CD4 (Additional file 4: Figure S3), IFNG (Additional file 5: Figure S4), IFNGR1 (Additional file 6: Figure S5), IFNGR2 (Additional file 7: Figure S6). For all these transcripts, EST alignment positions as well as protein domains are given. Furthermore, in order to identify the over-represented pathways in our AGM EST library, we performed a functional canonical pathway analysis based on the list of the 9,208 H. sapiens transcripts uniquely mapped by the 37,787 original ESTs. Most of the canonical pathways found as statistically significantly over-represented are related to B and T cell signaling, and immune response pathways (Table 4). For instance, the “CD28 signaling in T Helper Cells”, “iCOS-iCOSL signaling in T Helper Cells”, “B Cell receptor Signaling” (Additional file 8: Figure S7A), and “T Cell receptor signaling” (Additional file 8: Figure S7B) pathways belong to the list of pathways found as significantly over-represented in our AGM library, as well as the “Glucocorticoid receptor signaling”, “Role of NFAT in regulation of the immune response” (Additional file 9: Figure S8A), “Antigen presentation pathway” (Additional file 9: Figure S8B), “JAK/STAT signaling”, and many different “Interleukin signaling” pathways. As a result of the in vitro stimulation of SIV-infected PBMC, the “NF-κB Activation by viruses” (Additional file 10: Figure S9A) and “Induction of apoptosis by HIV-1” (Additional file 10: Figure S9B) pathways are also significantly over-represented. Consistent with the stimulation by LPS, the “Interferon Signaling” (Figure 5A) and “Toll-like Receptor Signaling” (Figure 5B) pathways are also found significantly over-represented. Finally, ConA is capable of triggering positive selection in mature T cells by cross-linking the TCR with high avidity [40,41] and we found 8 pathways corresponding to these functions being induced (Table 4). The over-representation of gene transcripts belonging to these pathways of the immune system further indicates that this library is a valuable resource for profiling global gene expression in AGM immune cells. Overall, these gene and pathway information are consistent with what we could expect from an EST PBMC library.

Bottom Line: For 506 transcripts, sequences were quasi-complete.In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes.Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut des Hautes Études Scientifiques-Centre National de la Recherche Scientifique, Bures-sur-Yvette, France.

ABSTRACT

Background: African Green Monkeys (AGM) are amongst the most frequently used nonhuman primate models in clinical and biomedical research, nevertheless only few genomic resources exist for this species. Such information would be essential for the development of dedicated new generation technologies in fundamental and pre-clinical research using this model, and would deliver new insights into primate evolution.

Results: We have exhaustively sequenced an Expression Sequence Tag (EST) library made from a pool of Peripheral Blood Mononuclear Cells from sixteen Chlorocebus sabaeus monkeys. Twelve of them were infected with the Simian Immunodeficiency Virus. The mononuclear cells were or not stimulated in vitro with Concanavalin A, with lipopolysacharrides, or through mixed lymphocyte reaction in order to generate a representative and broad library of expressed sequences in immune cells. We report here 37,787 sequences, which were assembled into 14,410 contigs representing an estimated 12% of the C. sabaeus transcriptome. Using data from primate genome databases, 9,029 assembled sequences from C. sabaeus could be annotated. Sequences have been systematically aligned with ten cDNA references of primate species including Homo sapiens, Pan troglodytes, and Macaca mulatta to identify ortholog transcripts. For 506 transcripts, sequences were quasi-complete. In addition, 6,576 transcript fragments are potentially specific to the C. sabaeus or corresponding to not yet described primate genes.

Conclusions: The EST library we provide here will prove useful in gene annotation efforts for future sequencing of the African Green Monkey genomes. Furthermore, this library, which particularly well represents immunological and hematological gene expression, will be an important resource for the comparative analysis of gene expression in clinically relevant nonhuman primate and human research.

Show MeSH
Related in: MedlinePlus