Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus.
Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus.
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DOI：
10.1186/1471-213x-7-69
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发表时间：
2007-06-18
影响因子：
--
通讯作者：
Manley NR
中科院分区：
文献类型：
--
作者：
Gordon J;Xiao S;Hughes B 3rd;Su DM;Navarre SP;Condie BG;Manley NR
胸腺上皮细胞（TEC）促进胸腺细胞成熟，是胸腺细胞发育早期和阳性选择所必需的。然而，由于缺乏遗传工具，对TEC执行这些功能的机制的研究受到抑制。由于Foxn 1基因在胸腺器官发生早期的所有推定TEC中表达，并且在成年胸腺中广泛表达，因此它是驱动分化和成熟TEC中基因表达的理想位点。我们通过将IRES-Cre或IRES-lacZ盒插入Foxn 1基因座的3' UTR中产生Foxn 1的两个敲入等位基因。我们同时电穿孔的两个靶向载体，以产生两个独立的等位基因在同一个实验中，证明了多重基因靶向在这个位点的可行性。我们的分析表明，敲入等位基因驱动表达Cre或lacZ在所有TEC在胎儿胸腺。此外，敲入等位基因以Foxn 1样模式表达Cre或lacZ，而不破坏Foxn 1功能，如通过Foxn 1敲入/Foxn 1无效复合杂合子的表型分析所确定的。这些数据表明，多重基因靶向Foxn 1基因座的3' UTR是从胸腺器官发生的最早阶段在TEC中表达任何感兴趣基因的有效方法。由此产生的等位基因将使TEC分化和功能的新的分子和遗传学研究成为可能。我们还讨论了表明基因靶向3' UTR是一种可广泛适用于产生遗传中性驱动菌株的技术的证据。
Thymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs. We generated two knock-in alleles of Foxn1 by inserting IRES-Cre or IRES-lacZ cassettes into the 3' UTR of the Foxn1 locus. We simultaneously electroporated the two targeting vectors to generate the two independent alleles in the same experiment, demonstrating the feasibility of multiplex gene targeting at this locus. Our analysis shows that the knockin alleles drive expression of Cre or lacZ in all TECs in the fetal thymus. Furthermore, the knockin alleles express Cre or lacZ in a Foxn1-like pattern without disrupting Foxn1 function as determined by phenotype analysis of Foxn1 knockin/Foxn1 null compound heterozygotes. These data show that multiplex gene targeting into the 3' UTR of the Foxn1 locus is an efficient method to express any gene of interest in TECs from the earliest stage of thymus organogenesis. The resulting alleles will make possible new molecular and genetic studies of TEC differentiation and function. We also discuss evidence indicating that gene targeting into the 3' UTR is a technique that may be broadly applicable for the generation of genetically neutral driver strains.