Практический подход к генетического картирования Судьба индуцибельной: Путеводитель по Марку и Трек Клетки В Vivo</em

Summary

Генетические индуцибельной Судьба картирования (GIFM) знаки и треков клеток с тонкой пространственной и временной контроль<em> В естественных условиях</em> И выясняет, как клетки с определенной генетической линии вклад в разработку и тканях взрослого организма. Продемонстрированный здесь приемы, необходимые для судьбы карте E12.5 эмбрионов мыши для epifluorescent и эксплантов анализа.

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 (CreER^T). CreER^T contains a modified estrogen receptor ligand binding domain which renders CreER^T sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreER^T, 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 CreER^T 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 CreER^T.

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-CreER^T;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

Тамоксифен Подготовка и устные зонд процедуры (E8.5) Подготовка 20 мг / мл исходного раствора тамоксифен, растворяя тамоксифен в подогретого кукурузное масло. Инкубируйте раствор при 37 ° С в течение 2 часов на nutator и вихревые периодически. Защита тамоксифен фондовом от свет…

Discussion

GIFM система, которую мы продемонстрировали, могут быть использованы для ответа широкий круг вопросов в различных клеточных процессов. Например, мыши разработана с различными драйверами Cre может быть использована для целевых любого типа клеток, тканей или генетической линии интересов. ?…