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Transcriptome analysis of mouse stem cells and early embryos.

Sharov AA, Piao Y, Matoba R, Dudekula DB, Qian Y, VanBuren V, Falco G, Martin PR, Stagg CA, Bassey UC, Wang Y, Carter MG, Hamatani T, Aiba K, Akutsu H, Sharova L, Tanaka TS, Kimber WL, Yoshikawa T, Jaradat SA, Pantano S, Nagaraja R, Boheler KR, Taub D, Hodes RJ, Longo DL, Schlessinger D, Keller J, Klotz E, Kelsoe G, Umezawa A, Vescovi AL, Rossant J, Kunath T, Hogan BL, Curci A, D'Urso M, Kelso J, Hide W, Ko MS - PLoS Biol. (2003)

Bottom Line: Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues.This can be a first step towards a possible definition of a molecular scale of cellular potency.The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Aging, Baltimore, Maryland, USA.

ABSTRACT
Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

Show MeSH
Examples of NIA-Only cDNA Clones and RT–PCR ResultsExpression pattern of 19 novel cDNA clones in 16 different cell lines or tissues: unfertilized egg, E3.5 blastocyst, E7.5 whole embryo (embryo plus placenta), E12.5 male mesonephros (gonad plus mesonephros), newborn brain, newborn ovary, newborn kidney, embryonic germ (EG) cell, embryonic stem (ES) cell (maintained as undifferentiated in the presence of LIF), trophoblast stem (TS) cell, mesenchymal stem (MS) cell, osteoblast, neural stem/progenitor (NS) cell, NS differentiated (differentiated neural stem/progenitor cells), and hematopoietic stem/progenitor (HS) cells. Glyceraldegyde-3-phosphate dehydrogenase (GAP-DH) was used as a control. A U number is assigned to each gene in the gene index (see Dataset S2). The exon number was predicted from alignment with the mouse genome sequence, and the amino acid sequence was predicted with the ORF finder from NCBI.
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pbio.0000074-g002: Examples of NIA-Only cDNA Clones and RT–PCR ResultsExpression pattern of 19 novel cDNA clones in 16 different cell lines or tissues: unfertilized egg, E3.5 blastocyst, E7.5 whole embryo (embryo plus placenta), E12.5 male mesonephros (gonad plus mesonephros), newborn brain, newborn ovary, newborn kidney, embryonic germ (EG) cell, embryonic stem (ES) cell (maintained as undifferentiated in the presence of LIF), trophoblast stem (TS) cell, mesenchymal stem (MS) cell, osteoblast, neural stem/progenitor (NS) cell, NS differentiated (differentiated neural stem/progenitor cells), and hematopoietic stem/progenitor (HS) cells. Glyceraldegyde-3-phosphate dehydrogenase (GAP-DH) was used as a control. A U number is assigned to each gene in the gene index (see Dataset S2). The exon number was predicted from alignment with the mouse genome sequence, and the amino acid sequence was predicted with the ORF finder from NCBI.

Mentions: Of 29,810 mouse genes identified in our gene index (Figure 1; Dataset S2; Dataset S3), 977 were not present as either known or predicted transcripts in other major transcriptome databases, such as RefSeq (Pruitt and Maglott 2001), Ensembl (Hubbard et al. 2002), and RIKEN (Okazaki et al. 2002) (see Dataset S3 for details and Dataset S4 for sequences). These genes represent possible novel mouse genes, as they either encode open reading frames (ORFs) greater than 100 amino acids or have multiple exons. In particular, 554 of the 977 genes remained novel with high confidence even after more thorough searches against GenBank and other databases. Comparisons of these 977 genes against all National Center for Biotechnology Information (NCBI) UniGene representative sequences showed that 377 genes did not match even fragmentary ESTs and are therefore unique to the National Institute on Aging (NIA) cDNA collection (see Dataset S3). A random subset of 19 cDNA clones representing these genes was sequenced completely to confirm their novelty (Figure 2). Protein domain searches using InterPro (Mulder et al. 2003) revealed that one of them, U004160, is an orthologue of human gene Midasin (MDN1), but the remaining 18 genes do not encode any known protein motifs. However, they were split into multiple exons in the alignment to the mouse genome sequences, and we therefore considered them genes. As these sequences are mainly derived from early embryos and stem cells, they most likely represent new candidates for genes specific to particular types of stem cells. RT–PCR analysis revealed that they are expressed in specific cell types (Figure 2; Dataset S5). For example, the expression of gene U035352 was unique to ES cells, expression of U004912 unique to ES and TS cells, and expression of U001905 unique to ES and EG cells. In addition, one gene showed apparent specific expression in several stem cells and is thus a potential pan-stem cell marker (U029765). Taken together, these data suggest that most of the putative genes represented only in the NIA cDNA collection are bona fide genes that have not been previously identified.


Transcriptome analysis of mouse stem cells and early embryos.

Sharov AA, Piao Y, Matoba R, Dudekula DB, Qian Y, VanBuren V, Falco G, Martin PR, Stagg CA, Bassey UC, Wang Y, Carter MG, Hamatani T, Aiba K, Akutsu H, Sharova L, Tanaka TS, Kimber WL, Yoshikawa T, Jaradat SA, Pantano S, Nagaraja R, Boheler KR, Taub D, Hodes RJ, Longo DL, Schlessinger D, Keller J, Klotz E, Kelsoe G, Umezawa A, Vescovi AL, Rossant J, Kunath T, Hogan BL, Curci A, D'Urso M, Kelso J, Hide W, Ko MS - PLoS Biol. (2003)

Examples of NIA-Only cDNA Clones and RT–PCR ResultsExpression pattern of 19 novel cDNA clones in 16 different cell lines or tissues: unfertilized egg, E3.5 blastocyst, E7.5 whole embryo (embryo plus placenta), E12.5 male mesonephros (gonad plus mesonephros), newborn brain, newborn ovary, newborn kidney, embryonic germ (EG) cell, embryonic stem (ES) cell (maintained as undifferentiated in the presence of LIF), trophoblast stem (TS) cell, mesenchymal stem (MS) cell, osteoblast, neural stem/progenitor (NS) cell, NS differentiated (differentiated neural stem/progenitor cells), and hematopoietic stem/progenitor (HS) cells. Glyceraldegyde-3-phosphate dehydrogenase (GAP-DH) was used as a control. A U number is assigned to each gene in the gene index (see Dataset S2). The exon number was predicted from alignment with the mouse genome sequence, and the amino acid sequence was predicted with the ORF finder from NCBI.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC300684&req=5

pbio.0000074-g002: Examples of NIA-Only cDNA Clones and RT–PCR ResultsExpression pattern of 19 novel cDNA clones in 16 different cell lines or tissues: unfertilized egg, E3.5 blastocyst, E7.5 whole embryo (embryo plus placenta), E12.5 male mesonephros (gonad plus mesonephros), newborn brain, newborn ovary, newborn kidney, embryonic germ (EG) cell, embryonic stem (ES) cell (maintained as undifferentiated in the presence of LIF), trophoblast stem (TS) cell, mesenchymal stem (MS) cell, osteoblast, neural stem/progenitor (NS) cell, NS differentiated (differentiated neural stem/progenitor cells), and hematopoietic stem/progenitor (HS) cells. Glyceraldegyde-3-phosphate dehydrogenase (GAP-DH) was used as a control. A U number is assigned to each gene in the gene index (see Dataset S2). The exon number was predicted from alignment with the mouse genome sequence, and the amino acid sequence was predicted with the ORF finder from NCBI.
Mentions: Of 29,810 mouse genes identified in our gene index (Figure 1; Dataset S2; Dataset S3), 977 were not present as either known or predicted transcripts in other major transcriptome databases, such as RefSeq (Pruitt and Maglott 2001), Ensembl (Hubbard et al. 2002), and RIKEN (Okazaki et al. 2002) (see Dataset S3 for details and Dataset S4 for sequences). These genes represent possible novel mouse genes, as they either encode open reading frames (ORFs) greater than 100 amino acids or have multiple exons. In particular, 554 of the 977 genes remained novel with high confidence even after more thorough searches against GenBank and other databases. Comparisons of these 977 genes against all National Center for Biotechnology Information (NCBI) UniGene representative sequences showed that 377 genes did not match even fragmentary ESTs and are therefore unique to the National Institute on Aging (NIA) cDNA collection (see Dataset S3). A random subset of 19 cDNA clones representing these genes was sequenced completely to confirm their novelty (Figure 2). Protein domain searches using InterPro (Mulder et al. 2003) revealed that one of them, U004160, is an orthologue of human gene Midasin (MDN1), but the remaining 18 genes do not encode any known protein motifs. However, they were split into multiple exons in the alignment to the mouse genome sequences, and we therefore considered them genes. As these sequences are mainly derived from early embryos and stem cells, they most likely represent new candidates for genes specific to particular types of stem cells. RT–PCR analysis revealed that they are expressed in specific cell types (Figure 2; Dataset S5). For example, the expression of gene U035352 was unique to ES cells, expression of U004912 unique to ES and TS cells, and expression of U001905 unique to ES and EG cells. In addition, one gene showed apparent specific expression in several stem cells and is thus a potential pan-stem cell marker (U029765). Taken together, these data suggest that most of the putative genes represented only in the NIA cDNA collection are bona fide genes that have not been previously identified.

Bottom Line: Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues.This can be a first step towards a possible definition of a molecular scale of cellular potency.The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Aging, Baltimore, Maryland, USA.

ABSTRACT
Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

Show MeSH