LongSAGE profiling of nine human embryonic stem cell lines.

To facilitate discovery of novel human embryonic stem cell (ESC) transcripts, we generated 2.5 million LongSAGE tags from 9 human ESC lines. Analysis of this data revealed that ESCs express proportionately more RNA binding proteins compared with terminally differentiated cells, and identified novel ESC transcripts, at least one of which may represent a marker of the pluripotent state.

Investigations of the genomic region that contains the clf1 mutation, a causal gene in multifactorial cleft lip and palate in mice.

BACKGROUND: Human nonsyndromic cleft lip and palate, CL(P), is genetically complex, with one contributing gene on chromosome 17q. A potentially homologous gene, clf1 on distal chromosome 11, is part of the digenic cause of the 10-30% CL(P) in the A/WySn mouse strain. Here we report our progress toward identifying the clf1 mutation. METHODS: Transcription from all of the known and predicted genes in the 1.5-Mb candidate region was examined in A/WySn and control (AXB-4/Pgn) ED10-11 embryo heads. The marker haplotype for 28 inbred strains across the clf1 region was obtained. The entire transcripts of Wnt9b and Wnt3 in A/WySn were sequenced. Using long PCR, the genomic region from Wnt3 throughWnt9b was screened in A/WySn for an inserted retrotransposon. RESULTS: Gosr2, Wnt9b, Wnt3, Nsf, Arf2, Crhr1, Mapt, Cdc27, Myl4, Itgb3, chr11_20.152, chr11_20.154, chr11_20.155, and chr11_20.156 are expressed in ED10-11 heads. None is absent or detectably reduced in A/WySn. The ancestral pre-clf1 mutation haplotype was found in CBA/J mice. By a test-cross, CBA/J was confirmed to lack the clf1 mutation. Three single-nucleotide variants in A/WySn (vs. C57BL/6J) were found in each of the 3' untranslated regions (3'UTRs) of Wnt3 and of Wnt9b, respectively; their presence in CBA/J shows that none are the clf1 mutation. An inserted intracisternal A particle (IAP) retrotransposon located 6.6 kb from the 3' end of Wnt9b was found in A/WySn and in all clf1 strains tested. This IAP is absent in C57BL/6J and CBA/J. CONCLUSIONS: The clf1 mutation is a genomic alteration present in A/WySn and absent in the ancestral chromosomal segment in CBA/J. The IAP retrotransposon insertion near Wnt9b in A/WySn fits this criterion; we predict that interference with Wnt9b function by this IAP is the clf1 mutation.

The open-eyelid mutation, lidgap-Gates, is an eight-exon deletion in the mouse Map3k1 gene.

The BALB/cGa mouse strain and its descendants, now called the SELH/Bc strain, have produced two waves of high frequency of spontaneous heritable mutations. One of these, the recessive lidgap-Gates (lg(Ga)) mutation, causes the same open-eyelids-at-birth phenotype as the gene knockout mutations of Map3k1 and co-maps to distal Chr 13. The lg(Ga) mutation is demonstrated to be a 27.5-kb deletion of exons 2-9 in the Map3k1 gene, the first spontaneous mutant allele described at this locus. The lg(Ga) mutation is consistent with a pattern suggesting that the waves of mutation in BALB/cGa and its descendants tend to be large deletions or ETn insertions, whose elevated rate of occurrence is due to an unknown mechanism.