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Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

ABSTRACT: The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.

SUBMITTER: Gadue P

PROVIDER: S-EPMC2890285 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

Gadue Paul P Gouon-Evans Valerie V Cheng Xin X Wandzioch Ewa E Zaret Kenneth S KS Grompe Markus M Streeter Philip R PR Keller Gordon M GM

Stem cells (Dayton, Ohio) 20090901 9

The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentra ...[more]

PMID: 19522011

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Project description:The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an indirect enzyme-linked immunosorbent assay for the detection of antibodies in sera from convalescent pigs showed no correlation with clinical and pathological findings.

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Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

Publications

Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

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