Gilchrist Derek S, Ure Jan, Hook Lilian, Medvinsky Alexander
Institute for Stem Cell Research, University of Edinburgh, West Main's Road, King's Buildings, Edinburgh, Scotland.
Genesis. 2003 Jul;36(3):168-76. doi: 10.1002/gene.10209.
Conditional activation and inactivation of genes using the Cre/loxP recombination system is a powerful tool for the analysis of gene function and for tracking cell fate. Here we report a novel silent EGFP reporter mouse line generated by enhancer trap technology using embryonic stem (ES) cells. Following transfection with the silent EGFP reporter construct, positive ES cell clones were treated with Cre recombinase. These "activated clones" were then further selected on the basis of ubiquitous EGFP expression during in vitro differentiation. The parental "silent" clones were then used for generating mice. Upon Cre-mediated activation in ovo tissues tested from these mice express EGFP. Long-term, strong and sustainable expression of EGFP is observed in most myeloid and lymphoid cells. As shown by in vivo transplantation assays, the majority of hematopoietic stem cells (HSCs) and spleen colony-forming units (CFU-S) reside within the EGFP positive fraction. Most in vitro colony-forming units (CFU-Cs) isolated from bone marrow also express EGFP. Thus, these reporter mice are useful for the analysis of Cre-mediated recombination in HSCs and hematopoietic progenitor cells. This, in combination with the high accessibility of the loxP sites, makes these mice a valuable tool for testing cell/tissue-specific Cre-expressing mice. .
利用Cre/loxP重组系统对基因进行条件性激活和失活，是分析基因功能和追踪细胞命运的有力工具。在此，我们报告一种通过增强子捕获技术利用胚胎干细胞（ES细胞）构建的新型沉默型EGFP报告基因小鼠品系。用沉默型EGFP报告基因构建体转染后，对阳性ES细胞克隆进行Cre重组酶处理。然后根据体外分化过程中普遍的EGFP表达情况进一步筛选这些“激活克隆”。接着用亲本“沉默”克隆来培育小鼠。在这些小鼠的卵内组织中经Cre介导激活后，所检测的组织表达EGFP。在大多数髓系和淋巴系细胞中观察到EGFP的长期、强烈且可持续的表达。体内移植试验表明，大多数造血干细胞（HSC）和脾集落形成单位（CFU-S）存在于EGFP阳性部分。从骨髓中分离出的大多数体外集落形成单位（CFU-C）也表达EGFP。因此，这些报告基因小鼠有助于分析HSC和造血祖细胞中Cre介导的重组。这与loxP位点的高可及性相结合，使这些小鼠成为测试细胞/组织特异性表达Cre的小鼠的有价值工具。
