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Regulatable in vivo biotinylation expression system in mouse embryonic stem cells.

ABSTRACT: Embryonic stem (ES) cells have several unique attributes, the two most important of which are they can differentiate into all cell types in the body and they can proliferate indefinitely. To study the regulation of these phenomena, we developed a regulatable in vivo biotinylation expression system in mouse ES cells. The E. coli biotin ligase gene BirA, whose protein product can biotinylate a 15-aa peptide sequence, called the AviTag, was cloned downstream of an IRES. The primary vector containing the doxycycline controlled transactivator gene tTA and IRES-BirA was knocked into the ROSA26 locus by homologous recombination. The secondary vector containing the AviTag tagged hKlf4 gene was exchanged into the ROSA26 locus using Cre recombinase. Western blot analysis showed that the doxycycline induced BirA protein can biotinylate the doxycycline induced AviTag tagged hKlf4 protein. The induction of hKlf4 repressed cell growth in the presence or absence of LIF. Chromatin immunoprecipitation assays using streptavidin beads showed that the AviTag tagged hKlf4 protein could enrich the Nanog enhancer. Our results suggested that the regulatable biotinylation system is promising for the gene function studies in mouse ES cells.

SUBMITTER: Wang Q

PROVIDER: S-EPMC3646753 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Regulatable in vivo biotinylation expression system in mouse embryonic stem cells.

Wang Qin Q Wagner Ryan T RT Cooney Austin J AJ

PloS one 20130507 5

Embryonic stem (ES) cells have several unique attributes, the two most important of which are they can differentiate into all cell types in the body and they can proliferate indefinitely. To study the regulation of these phenomena, we developed a regulatable in vivo biotinylation expression system in mouse ES cells. The E. coli biotin ligase gene BirA, whose protein product can biotinylate a 15-aa peptide sequence, called the AviTag, was cloned downstream of an IRES. The primary vector containin ...[more]

PMID: 23667633

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Project description:BackgroundUnderstanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1-4 EBs.ResultsAn initial array study identified 4 gene expression changes between 3 undifferentiated ES cell lines. Tissue culture conditions for ES differentiation were then optimized to give the maximum range of gene expression and growth. -Undifferentiated ES cells and EBs cultured with and without LIF at each day for 4 days were subjected to microarray analysis. -Differential expression of 23 genes was identified. 13 of these were also differentially regulated in a separate array comparison between undifferentiated ES cells and compartments of very early embryos. A high degree of inter-replicate variability was noted when confirming array results. Using a panel of marker genes, RNA amplification and RT-PCR, we examined expression pattern variation between individual -D4-Lif EBs. We found that individual EBs selected from the same dish were highly variable in gene expression profile.ConclusionES cell lines derived from different mouse strains and carrying different genetic modifications are almost invariant in gene expression profile under conditions used to maintain pluripotency. Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these are also identified using similar studies, thus validating our results. EBs cultured in the same dish vary widely in terms of their gene expression (and hence, undoubtedly, in their future differentiation potential). This may explain some of the inherent variability in differentiation protocols that use EBs.

Dataset Information

Regulatable in vivo biotinylation expression system in mouse embryonic stem cells.

Publications

Regulatable in vivo biotinylation expression system in mouse embryonic stem cells.

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