A conditional floxed (IoxP-flanked) allele for the retinoic acid receptor beta (RARβ) gene
A conditional floxed (IoxP-flanked) allele for the retinoic acid receptor beta (RARβ) gene
复制标题
DOI：
10.1002/gene.10073
复制
发表时间：
2002-02-01
期刊：
影响因子：
1.5
通讯作者：
Ghyselinck, NB
中科院分区：
文献类型：
--
作者：
Chapellier, B;Mark, M;Ghyselinck, NB
视黄酸（RA）通过与核受体（RAR/RXR异二聚体）结合而起作用，所述核受体属于配体诱导型转录因子超家族（Chambon，1996）。为了鉴定三种RAR同种型（α、β和γ）在体内类维生素A信号传导途径中的功能，已经破坏了它们的基因。缺乏一种RAR同种型的小鼠只显示出生后维生素A缺乏症（VAD）引起的一些缺陷，而在两种RAR同种型的复合无效突变体中发现了重现胎儿VAD综合征的异常。这些发现得出的结论是：（i）RAR参与大多数组织的形态发生和器官发生的多个阶段，（ii）在许多情况下，功能冗余允许（可能是人为的）剩余的RAR弥补缺失的RAR。所有的复合无效突变体在出生时或胚胎发育期间的早期死亡，排除了在后期阶段对RAR功能的分析（Kastner等人，1995，1997; Mascrez等人，1998; Mark等人，1999年;参考文献）。因此，RAR在发育过程中和成人中的大部分生理功能仍然未知。此外，生殖系突变不足以区分RAR的细胞自主和非细胞自主功能。它们的阐明将需要时空控制的体细胞突变。在这方面，基于使用他莫昔芬诱导型Cre-ERT重组酶的条件诱变方法应允许RAR基因在任何给定时间和在选定的细胞类型中失活（Metzger和Chambon，2001）。为此，我们报告了在RARβ基因座处具有“floxed”（loxP侧翼）条件等位基因的小鼠系的产生。靶向载体包括外显子8-11（E8-E11，氨基酸残基204-448; Zelent等人，1989），并含有外显子8上游的loxP侧翼的新霉素选择基因，以及外显子10下游的loxP（图1A）。在胚胎干（ES）细胞中同源重组后，通过Southern印迹法鉴定了6个含有loxP侧翼L3 RARβ等位基因的阳性克隆（共278个）（图1B）。用编码pSG 5Cre的质粒电穿孔这些克隆之一（NE 75）（JM Garnier和PC，未发表的数据）。在86个亚克隆中分析了Cre介导的重组。在一些ES细胞中，整个loxP侧翼的DNA片段被切除（RARβ Lj等位基因），而在6例病例中，仅新霉素盒被切除（floxed L2等位基因，例如，亚克隆NE 75）。4;图1C）。源自亚克隆NE 75的嵌合雄性。4通过其生殖系传递RARβ L2等位基因，产生floxed RARβL2/L2小鼠系。携带floxed L2等位基因的小鼠表达正常水平的RARβ，并且与野生型同窝出生的小鼠无法区分（图2B、C，数据未显示）。为了检查在RARβ基因座处Cre介导的切除是否可以在体内发生，将RARβL2/L2小鼠与在生殖细胞中表达Cre重组酶的CMV-Rg/0转基因小鼠杂交（Dupé等人，1997年）。尾DNA的PCR分析显示Cre依赖性RARβ L2等位基因切除，产生RARβ+/LJ杂合动物（图2A）。切除的Lj等位基因携带编码大部分配体结合结构域的外显子9和10的缺失（氨基酸残基263-384; Zelent等人，1989年），并导致移码突变。为了验证是否产生无效等位基因，将RARβ+/LJ小鼠杂交以产生RARβLJ/LJ纯合子，其纹状体不表达RARβ蛋白（图2B）。RARβLJ/LJ成年小鼠的眼切片显示玻璃体内存在色素组织的异常晶状体后团块（R，比较图2C……）
Retinoic acid (RA) acts through binding to nuclear receptors (RAR/RXR heterodimers), which belong to the ligand-inducible transcription factors superfamily (Chambon, 1996). To identify the functions of the three RAR isotypes (α, β, and γ) in the retinoid signaling pathway in vivo, their genes have been disrupted. Mice lacking one RAR isotype display only some of the defects resulting from postnatal vitamin A deficiency (VAD), whereas abnormalities recapitulating the fetal VAD syndrome were found in compound null mutants for two RAR isotypes. These findings led to the conclusion that (i) RARs are involved at multiple stages during morphogenesis and organogenesis of most tissues and (ii) in many instances functional redundancy allow, possibly artefactually, the remaining RARs to compensate for the missing one. All the compound null mutants die at birth or earlier during embryonic development, precluding analysis of RAR functions at later stages (Kastner et al., 1995, 1997; Mascrez et al., 1998; Mark et al., 1999; references therein). Thus, most of the physiological functions of RARs during development and in adults remain unknown. Additionally, germ-line mutations are inadequate to discriminate between cell-autonomous and non-cell-autonomous functions of RARs. Their elucidation will require spatio-temporally controlled somatic mutations. In this regard, the conditional mutagenesis approach based on the use of tamoxifen-inducible Cre-ERT recombinases should permit the inactivation of the RAR genes at any given time and in a chosen cell type (Metzger and Chambon, 2001). To this end, we report the generation of a mouse line harbouring a “floxed”(loxP-flanked) conditional allele at the RARβ locus. The targeting vector encompasses exons 8–11 (E8–E11, amino-acid residues 204–448; Zelent et al., 1989) and contains a loxP-flanked neomycin selector gene upstream of exon 8, as well as a loxP downstream of exon 10 (Fig. 1A). After homologous recombination in embryonic stem (ES) cells, six positive clones (out of 278) containing a loxP-flanked L3 RARβ allele were identified by Southern blotting (Fig. 1B). One of these clones (NE75) was electroporated with a pSG5Cre-encoding plasmid (JM Garnier and PC, unpublished data). Cremediated recombination was analyzed in 86 subclones. In some ES cells, the entire loxP-flanked DNA fragment was excised (RARβ Lj allele), whereas in 6 cases the neomycin cassette only was excised (floxed L2 allele, eg, subclone NE75. 4; Fig. 1C). Chimeric males derived from subclone NE75. 4 transmitted the RARβ L2 allele through their germ line, yielding the floxed RARβL2/L2 mouse line. Mice carrying the floxed L2 alleles expressed normal levels of RARβ and were indistinguishable from wild-type littermates (Fig. 2B, C, and data not shown). To check whether Cre-mediated excision at the RARβ locus can occur in vivo, RARβL2/L2 mice were crossed with CMV-Cretg/0 transgenic mice that express the Cre recombinase in germ cells (Dupé et al., 1997). PCR analysis of tail DNA revealed a Cre-dependent RARβ L2 allele excision, yielding RARβ+/LJ heterozygous animals (Fig. 2A). The excised Lj allele bears a deletion of exons 9 and 10 that encodes most of the ligand-binding domain (amino-acid residues 263–384; Zelent et al., 1989) and causes a frameshift mutation. To verify that a null allele was generated, RARβ+/LJ mice were intercrossed to produce RARβLJ/LJ homozygotes whose striatum did not express the RARβ protein (Fig. 2B). Eye sections from RARβLJ/LJ adult mice revealed the presence within the vitreous body of an abnormal retrolenticular mass of pigmented tissue (R, compare Fig. 2C …