Development of a bovine X chromosome linkage group and painting probes to assess cattle, sheep, and goat X chromosome segment homologies

The X chromosome linkage group is conserved in placental mammals. However, X chromosome morphological differences, due to internal chromosome rearrangements, exist among mammalian species. We have developed bovine chromosome painting probes for Xp and Xq to assess segment homologies between the submetacentric bovine X chromosome and the acrocentric sheep and goat X chromosomes. These painting probes and their corresponding DNA libraries were developed by chromosome micromanipulation, DNA micropurification, microcloning, and PCR amplification. The bovine Xp painting probe identified an interstitially located homologous segment in the sheep and goat Xq region, most probably resulting from chromosome inversion. Ten type II (microsatellite) markers obtained from the bovine Xq library and five other X chromosome assigned, but unlinked, markers were used to generate a linkage map for Xq spanning 89.4 centimorgans. The chromosome painting probes and molecular markers generated in this study would be useful for comparative mapping and tracing of internal X chromosome rearrangements in all ruminant species and would contribute to the understanding of mammalian sex chromosome evolution

Molecular approaches for the construction of integrated physical and genetic maps

This study reports the development of chromosome-specific libraries by chromosome microdissection and microcloning and its utility in developing high density linkage map for particular chromosomes. Chromosome-specific painting probes were prepared for bovine (Bos taurus) autosomes 11 and 23 using two different translocation cell lines. Chromosome painting probe for swine chromosome 6 was developed using chromosomes from primary swine fibroblast cultures. The purity and specificity of the painting probes was verified by fluorescent in situ hybridization (FISH) on bovine and swine metaphase chromosomes. Bovine painting probes were used on sheep (Ovis aries) and goat (Capra hircus) metaphases to identify their corresponding homologs. BTA 11 and SSA 6-specific DNA fragments were cloned in Lambda Zap Express vector to develop high titer chromosome-specific libraries. BTA 11 library was screened for microsatellite-containing clones using (AC)\sb{12} oligos. Primer pairs developed for 17 microsatellites yielded successful amplifications with bovine genomic DNA. Three markers were binned on Illinois Reference/Resource family (IRRF) and 14 were mapped on the USDA-MARC resource family. Two point analysis was done on MARC population to generate a preliminary linkage map for BTA 11. A bovine YAC library was screened with BTA 11-specific microsatellite primers. Four YACs were identified and physically mapped by FISH. Two YAC clones that were mapped to BTA 11 by linkage and by FISH helped to orient and anchor the linkage map on bovine chromosome 11. BTA 11-specific DNA was subjected to subtractive hybridization using bovine Cot4 DNA and the subtracted product was used to screen a bovine cosmid library. Positive clones were pooled and mapped to bovine metaphases by FISH. All the clones showed very strong hybridization on the centromeres of all autosomes in the bovine chromosome complement. However, they failed to hybridize to the sex chromosome centromeres suggesting that the sequences are specific to autosomal centromeres. The same probes failed to hybridize to sheep and goat metaphases suggesting species specificity of these probes. An answer to the exact function of these DNA sequences need to be investigated

FGF2-induced effects on transcriptome associated with regeneration competence in adult human fibroblasts

BACKGROUND: Adult human fibroblasts grown in low oxygen and with FGF2 supplementation have the capacity to tip the healing outcome of skeletal muscle injury – by favoring regeneration response in vivo over scar formation. Here, we compare the transcriptomes of control adult human dermal fibroblasts and induced regeneration-competent (iRC) fibroblasts to identify transcriptional changes that may be related to their regeneration competence. RESULTS: We identified a unique gene-expression profile that characterizes FGF2-induced iRC fibroblast phenotype. Significantly differentially expressed genes due to FGF2 treatment were identified and analyzed to determine overrepresented Gene Ontology terms. Genes belonging to extracellular matrix components, adhesion molecules, matrix remodelling, cytoskeleton, and cytokines were determined to be affected by FGF2 treatment. CONCLUSIONS: Transcriptome analysis comparing control adult human fibroblasts with FGF2-treated fibroblasts identified functional groups of genes that reflect transcriptional changes potentially contributing to their regeneration competence. This comparative transcriptome analysis should contribute new insights into genes that characterize cells with greater regenerative potential

RT-PCR for expression of embryonic stem cell specific genes.

<p>OCT4, NANOG and CRIPTO1 in human ESCs during culture (A), during 21 days after induction of hESC differentiation (B), and in adult human dermal fibroblasts cultured in 2% oxygen with FGF2 supplementation (C). Restriction digest of 646 bp OCT4 amplicon from human embryonic stem cells (D), control fibroblasts (E), and iPSCs (F). ApaI restriction digest of OCT4 amplicon in fibroblasts grown in 2% oxygen and FGF2 supplementation (G), and various transformed, multipotent and differentiated cells (H). NCCIT – teratocarcinoma, NTERA2– teratocarcinoma, SHSY – neuroblastoma, SMCs – smooth muscle cells, HUVECs – human umbilical vein endothelial cells, hMSCs – human mesenchymal stem cells. Only the embryonic OCT4A contains ApaI restriction site.</p

Restriction digest of 646 bp OCT4 amplicon from human embryonic carcinoma cells during 21 days of differentiation in medium containing retinoic acid.

<p>Restriction digest of 646 bp OCT4 amplicon from human embryonic carcinoma cells during 21 days of differentiation in medium containing retinoic acid.</p

Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells

<div><p>The POU5F1 gene codes for the OCT4 transcription factor, which is one of the key regulators of pluripotency. Its transcription, alternative splicing, and alternative translation leading to the synthesis of the active, nuclear localized OCT4A has been described in detail. Much less, however, is known about actively transcribed OCT4 pseudogenes, several of which display high homology to OCT4A and can be expressed and translated into proteins. Using RT-PCR followed by pseudogene-specific restriction digestion, cloning, and sequencing we discriminate between OCT4A and transcripts for pseudogenes 1, 3 and 4. We show that expression of OCT4 and its pseudogenes follows a developmentally-regulated pattern in differentiating hESCs, indicating a tight regulatory relationship between them. We further demonstrate that differentiated human cells from a variety of tissues express exclusively pseudogenes. Expression of OCT4A can, however be triggered in adult differentiated cells by oxygen and FGF2-dependent mechanisms.</p></div