Brown Ashly, Brown Stephen, Ellisor Debra, Hagan Nellwyn, Normand Elizabeth, Zervas Mark
Department of Neuroscience, Division of Biology and Medicine, Brown University, USA.
J Vis Exp. 2009 Dec 30(34):1687. doi: 10.3791/1687.
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.
命运图谱是通过在体内标记和追踪细胞来生成的，以确定祖细胞如何在发育中的组织和成年组织中形成特定结构和细胞类型。这一概念的一个进展是遗传诱导命运图谱（GIFM），它将体内的基因表达、细胞命运和细胞行为联系起来，以创建基于遗传谱系的命运图谱。GIFM利用X-CreER系，其中X是一个基因或一组基因调控元件，可赋予修饰的噬菌体蛋白Cre重组酶（CreER(T)）空间表达。CreER(T)包含一个修饰的雌激素受体配体结合域，在没有药物他莫昔芬的情况下，CreER(T)被隔离在细胞质中。他莫昔芬的结合会释放CreER(T)，后者转移到细胞核并介导loxP位点侧翼的DNA序列之间的重组。在GIFM中，重组通常发生在报告基因（如GFP）之前的loxP侧翼的终止盒之间。培育含有区域或细胞类型特异性CreER和条件报告等位基因的小鼠。未处理的小鼠不会有标记，因为报告基因中的终止盒会阻止报告基因的进一步转录。我们通过口服灌胃法给定时怀孕的雌性小鼠施用他莫昔芬，这提供了对CreER(T)释放以及随后转移到细胞核从而从报告基因中去除终止盒的时间控制。重组后，报告等位基因会持续且可遗传地表达。这一系列事件标记了细胞，使其遗传历史被永久记录。因此，重组后的报告基因作为一种高保真的遗传谱系示踪剂，一旦开启，就与最初用于驱动CreER(T)的基因表达脱钩。我们在小鼠中应用GIFM来研究正常发育，并确定遗传谱系对成年细胞类型和组织的贡献。我们还使用GIFM追踪突变遗传背景下的细胞，以更好地理解模拟人类遗传疾病显著特征的复杂表型。这篇视频文章指导研究人员通过实验方法成功应用GIFM。我们通过在胚胎第8.5天（E）通过口服灌胃施用他莫昔芬，然后在E12.5进行解剖并通过落射荧光立体显微镜分析，使用我们特征明确的Wnt1-CreER(T);mGFP小鼠来演示该方法。我们还演示了如何显微解剖命运图谱区域以进行外植体制备或流式细胞术分析，以及解剖成年命运图谱大脑以进行整体荧光成像。总的来说，这些程序使研究人员能够解决发育生物学和疾病模型中的关键问题。
