जेनेटिक inducible किस्मत मानचित्रण के लिए एक व्यावहारिक दृष्टिकोण: एक दृश्य गाइड मार्क और ट्रैक कक्ष Vivo में</em
Summary
जेनेटिक inducible किस्मत मानचित्रण (GIFM) के निशान और ठीक स्थानिक और लौकिक नियंत्रण के साथ पटरियों कोशिकाओं<em> Vivo में</em> Elucidates और कैसे एक विशिष्ट आनुवंशिक वंश से कोशिकाओं के विकास और वयस्क ऊतकों के लिए योगदान. यहाँ प्रदर्शन कर रहे हैं भाग्य का नक्शा E12.5 epifluorescent और explant विश्लेषण के लिए माउस भ्रूण के लिए आवश्यक तकनीक हैं.
Abstract
Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.
Protocol
Discussion
GIFM प्रणाली है कि हम दिखा दिया है करने के लिए सेलुलर प्रक्रियाओं की एक किस्म में सवालों की एक विस्तृत श्रृंखला का जवाब करने के लिए इस्तेमाल किया जा सकता है. उदाहरण के लिए, अलग Cre चालकों के साथ इंजीनियर चूहो?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
हम mGFP चूहों के लिए एस Arber और Zervas प्रयोगशाला जो गंभीर अखबार पढ़ा और तकनीकी सहायता प्रदान की सदस्यों को आभारी हैं. ब्राउन एक मस्तिष्क विज्ञान कापलान ग्रीष्मकालीन स्नातक अनुसंधान पुरस्कार द्वारा समर्थित किया गया. ई. नोरमंड एक मस्तिष्क विज्ञान कार्यक्रम ग्रेजुएट अनुसंधान पुरस्कार द्वारा समर्थित किया गया था. इस शोध भी स्टार्टअप अनुसंधान कोष (MZ) द्वारा वित्त पोषित किया गया था.
Riferimenti
- Ellisor, D., Koveal, D., Hagan, N., Brown, A., Zervas, M. Comparative analysis of conditional reporter alleles in the developing embryo and embryonic nervous system. Gene Expr Patterns. 9, 475-489 (2009).
- Zervas, M., Millet, S., Ahn, S., Joyner, A. L. Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1. Neuron. 43, 345-357 (2004).
