Name: a practical approach to genetic inducible fate mapping: a visual guide to mark and track cells in vivo
Uploaded: 2009-12-30T15:00:00
Description: Watch this Scientific Journal Video about A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo at JoVE.com

We are grateful to S. Arber for the mGFP mice and to members of the Zervas lab who critically read the paper and provided technical assistance. A. Brown was supported by a Brain Science Kaplan Summer Graduate Research Award. E. Normand was supported by a Brain Science Program Graduate Research Award. This research was also funded by startup research funds (MZ).

Tamoxifen Preparation and Oral Gavage Procedure (E8.5)
<ul>
 <li> Prepare a 20 mg/ml stock solution of tamoxifen by dissolving tamoxifen in pre-warmed corn oil. </li>
 <li> Incubate the solution at 37&deg; C for 2 hours on a nutator and vortex intermittently.</li>
 <li> Protect the tamoxifen stock from light, store at 4&deg;C, and use for up to one month. </li>
 <li> For fate mapping experiments, establish a breeding pair consisting of a Swiss Webster female (wildtype; purchased from Taconic) and a...

<ol>
	<li>Ellisor, D., Koveal, D., Hagan, N., Brown, A., Zervas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+conditional+reporter+alleles+in+the+developing+embryo+and+embryonic+nervous+system.">Comparative analysis of conditional reporter alleles in the developing embryo and embryonic nervous system.</a> Gene Expr Patterns. 9, 475-489 (2009).</li><li>Zervas, M., Millet, S., Ahn, S., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+behaviors+and+genetic+lineages+of+the+mesencephalon+and+rhombomere+1.">Cell behaviors and genetic lineages of the mesencephalon and rhombomere 1.</a> Neuron. 43, 345-357 (2004).</li></ol>

The GIFM system that we have demonstrated can be used to answer a wide range of questions in a variety of cellular processes. For example, mice engineered with different Cre drivers can be used to target any cell type, tissue or genetic lineage of interest. In addition, because tamoxifen can be administered at any embryonic or postnatal time point, recombination can be targeted to any relevant developmental stage. The spatial and temporal control of this system are ideal for investigating how cells expressing particular...

a practical approach to genetic inducible fate mapping: a visual guide to mark and track cells in vivo

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.

Watch this Scientific Journal Video about A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo at JoVE.com

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

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