हम एक chromatin immunoprecipitation (चिप) विधि प्रदर्शित करने के लिए माउस भ्रूण ऊतक में ऊतक विशिष्ट जीन की अभिव्यक्ति की शुरुआत के दौरान या बाद में ऊतक विशिष्ट जीन में कारक बातचीत की पहचान. इस प्रोटोकॉल व्यापक रूप से ऊतक विशिष्ट जीन सक्रियण के अध्ययन के लिए लागू हो सकता है के रूप में यह सामान्य भ्रूण विकास के दौरान होता है चाहिए.
Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.
वर्णित चिप प्रोटोकॉल में, हम बताते हैं कि myogenic नियामक myogenin कंकाल की मांसपेशी अग्रदूत एकल E8.5 और E9.5 भ्रूण में मौजूद ऊतकों में myogenin प्रमोटर के साथ जुड़ा हुआ है. पूर्व अध्ययनों से बड़े पैमाने पर ई बॉक्स युक्त दृश?…
The authors have nothing to disclose.
इस काम आईएएनएस, जो अमेरिकी रिकवरी और पुनर्निवेश अधिनियम 2009 के माध्यम से सम्मानित किया धन शामिल R01 GM56244 NIH द्वारा समर्थित किया गया था, और R01 GM87130 NIH द्वारा JARP
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
ChIP Assay Kit | Upstate Cell Signaling Solutions, Millipore | 17-295 | ||
Collagenase Type II | Invitrogen | 17101015 | Dilution by 1 x PBS | |
Dulbecco’s modified eagle medium (DMEM) | Gibco Labs, Invitrogen | 12100-061 | High glucose content | |
Dulbecco’s phosphate buffered saline 1X (DPBS) | Gibco Labs, Invitrogen | 14190-144 | Calcium chloride free, Magnesium chloride free | |
Fetal bovine serum (FBS) | Mediatech, Inc. | 35-010-CV | ||
Gel extraction kit | QIAquick | 28704 | 50 reaction kit | |
Penicillin/streptomycin stock solution | Gibco Labs, Invitrogen | 5000 μg/ml concentration | ||
Protease Inhibitor Cocktail | Sigma-Aldrich | P8340 | ||
Salmon sperm DNA /Protein A agarose | Millipore | 16-157 | ||
myogenin antibody | Santa Cruz Biotechnology, Inc. | sc-576 | ||
Normal rabbit IgG | Millipore | 12-370 | ||
Platinum PCR Supermix | Invitrogen | 11306-016 | ||
GoTaq Q-PCR master mix | Promega | A6001 |