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From RNAi screens to molecular function in embryonic stem cells.

Ding L, Poser I, Paszkowski-Rogacz M, Buchholz F - Stem Cell Rev (2012)

Bottom Line: Fueled by this interest, intense research has provided new insights into the biology of ES cells in the recent past.The development of large-scale and high throughput RNAi technologies has made it possible to sample the role of every gene in maintaining ES cell identity.Furthermore, we provide a perspective on how to streamline the molecular characterization following the initial phenotypic description utilizing bacterial artificial chromosome (BAC) transgenesis.

View Article: PubMed Central - PubMed

Affiliation: University Hospital Carl Gustav Carus and Medical Faculty, University of Technology Dresden, Fetscherstr. 74, 01307, Dresden, Germany.

ABSTRACT
The ability of embryonic stem (ES) cells to generate any of the around 220 cell types of the adult body has fascinated scientists ever since their discovery. The capacity to re-program fully differentiated cells into induced pluripotent stem (iPS) cells has further stimulated the interest in ES cell research. Fueled by this interest, intense research has provided new insights into the biology of ES cells in the recent past. The development of large-scale and high throughput RNAi technologies has made it possible to sample the role of every gene in maintaining ES cell identity. Here, we review the RNAi screens performed in ES cells to date and discuss the challenges associated with these large-scale experiments. Furthermore, we provide a perspective on how to streamline the molecular characterization following the initial phenotypic description utilizing bacterial artificial chromosome (BAC) transgenesis.

Show MeSH
Immunostaining of mouse ES cells stably expressing GFP-tagged transgenes. a AURKB, b Ki67. Green: GFP-tagged transgene; Red: alpha-tubulin; Blue: DAPI (scale bar: 10 μm)
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Fig5: Immunostaining of mouse ES cells stably expressing GFP-tagged transgenes. a AURKB, b Ki67. Green: GFP-tagged transgene; Red: alpha-tubulin; Blue: DAPI (scale bar: 10 μm)

Mentions: Determination of protein localization within cells is a vital and frequently used method to characterize a protein of interest including assumptions on its potential role in protein pathways and networks. Antibodies are typically used to investigate the localization of proteins. However, their generation is cost-intensive and time-consuming and the optimal staining protocol for each antibody is different and has to be optimized. Furthermore, antibody staining does not allow for a spatio-temporal investigation of protein localization. The generation of BAC-tagged GFP fusion proteins offers a versatile approach that overcomes many of these limitations. First, cell lines expressing BAC-tagged GFP fusion proteins can be rapidly and cost-effectively generated. Importantly, the fusion protein is typically expressed near physiological levels, avoiding localization artifacts often observed when GFP-fusion proteins are over-expressed from cDNA constructs. Second, a single, well-defined antibody against GFP can be used employing the same staining protocol for all tagged proteins (Fig. 5). Third, following the GFP signal in live cells can provide valuable spatio-temporal information of protein localization such as protein behavior during the cell cycle or during cell differentiation.Fig. 5


From RNAi screens to molecular function in embryonic stem cells.

Ding L, Poser I, Paszkowski-Rogacz M, Buchholz F - Stem Cell Rev (2012)

Immunostaining of mouse ES cells stably expressing GFP-tagged transgenes. a AURKB, b Ki67. Green: GFP-tagged transgene; Red: alpha-tubulin; Blue: DAPI (scale bar: 10 μm)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285751&req=5

Fig5: Immunostaining of mouse ES cells stably expressing GFP-tagged transgenes. a AURKB, b Ki67. Green: GFP-tagged transgene; Red: alpha-tubulin; Blue: DAPI (scale bar: 10 μm)
Mentions: Determination of protein localization within cells is a vital and frequently used method to characterize a protein of interest including assumptions on its potential role in protein pathways and networks. Antibodies are typically used to investigate the localization of proteins. However, their generation is cost-intensive and time-consuming and the optimal staining protocol for each antibody is different and has to be optimized. Furthermore, antibody staining does not allow for a spatio-temporal investigation of protein localization. The generation of BAC-tagged GFP fusion proteins offers a versatile approach that overcomes many of these limitations. First, cell lines expressing BAC-tagged GFP fusion proteins can be rapidly and cost-effectively generated. Importantly, the fusion protein is typically expressed near physiological levels, avoiding localization artifacts often observed when GFP-fusion proteins are over-expressed from cDNA constructs. Second, a single, well-defined antibody against GFP can be used employing the same staining protocol for all tagged proteins (Fig. 5). Third, following the GFP signal in live cells can provide valuable spatio-temporal information of protein localization such as protein behavior during the cell cycle or during cell differentiation.Fig. 5

Bottom Line: Fueled by this interest, intense research has provided new insights into the biology of ES cells in the recent past.The development of large-scale and high throughput RNAi technologies has made it possible to sample the role of every gene in maintaining ES cell identity.Furthermore, we provide a perspective on how to streamline the molecular characterization following the initial phenotypic description utilizing bacterial artificial chromosome (BAC) transgenesis.

View Article: PubMed Central - PubMed

Affiliation: University Hospital Carl Gustav Carus and Medical Faculty, University of Technology Dresden, Fetscherstr. 74, 01307, Dresden, Germany.

ABSTRACT
The ability of embryonic stem (ES) cells to generate any of the around 220 cell types of the adult body has fascinated scientists ever since their discovery. The capacity to re-program fully differentiated cells into induced pluripotent stem (iPS) cells has further stimulated the interest in ES cell research. Fueled by this interest, intense research has provided new insights into the biology of ES cells in the recent past. The development of large-scale and high throughput RNAi technologies has made it possible to sample the role of every gene in maintaining ES cell identity. Here, we review the RNAi screens performed in ES cells to date and discuss the challenges associated with these large-scale experiments. Furthermore, we provide a perspective on how to streamline the molecular characterization following the initial phenotypic description utilizing bacterial artificial chromosome (BAC) transgenesis.

Show MeSH