A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells
A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells
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DOI：
10.1093/nar/23.24.5080
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发表时间：
1995-12-25
影响因子：
14.9
通讯作者：
Rajewsky, K
中科院分区：
文献类型：
--
作者：
Schwenk, F;Baron, U;Rajewsky, K
利用噬菌体P1 Cre-loxP重组系统，研究小鼠基因功能的基因靶向方法最近得到了改进。Cre重组酶催化34个bprecognition （loxP）位点之间的位点特异性DNA重组（1,2）。如果两个loxP位点以相同的方向引入基因组位点，Cre的表达会导致loxP侧的DNA序列缺失。cre介导的重组可以应用于胚胎干（ES）细胞中的各种类型的基因操作，包括去除可选标记基因并引入细微突变(3)，基因替换(4)以及删除大基因组区域以使基因或基因簇失活(5)。然而，Cre介导的基因操作需要用载体编码Cre重组酶瞬时转染含有loxp的突变ES细胞，并进行额外的筛选，以筛选携带所需缺失的ES细胞克隆。这种对培养细胞的额外操作是令人不快的，因为它可能会影响胚胎干细胞在嵌合小鼠中产生生殖细胞的能力，这是基因靶向技术中的一个严重问题。Cre-loxP重组系统也被用于以细胞类型特异性（6-8）或诱导(9)的方式操纵基因。如果将两个loxP位点引入基因组，使它们位于靶基因的重要部分的两侧而不影响其功能，则cre介导的loxP侧翼片段的缺失将导致该基因失活。在细胞类型特异性或诱导启动子的控制下，通过将带有loxp侧翼靶基因的小鼠与表达cre的转基因小鼠杂交，可以将体内基因失活限制在特定的细胞类型或时间点。这类实验通常涉及培养两种独立的小鼠品系，一种含有loxp -基因，适合条件失活，另一种缺失相应的基因，以研究基因在小鼠发育过程中的功能。然而，这需要将不同的突变ES细胞克隆注射到囊胚中以产生两个独立的小鼠品系，这是昂贵且耗时的。这些问题的Cre-loxP介导的基因靶向可以很大程度上克服在我们已经产生的转基因小鼠品系的帮助下。该菌株介导包括生殖细胞在内的所有组织中loxp -侧翼基因的缺失（图1）。该菌株是通过注射DNA片段产生的
The gene targeting approach to study gene function in mice has been recently refined using the bacteriophage P1 Cre-loxP recombination system. Cre recombinase catalyses site-specific DNA recombination between 34 bprecognition (loxP) sites (1, 2). If two loxP sites are introduced in the same orientation into a genomic locus, expression of Cre results in the deletion of the loxP-flanked DNA sequence. Cre-mediated recombinationcan be applied to various types of gene manipulation in embryonic stem (ES) cells including the removal ofaselectable marker gene accompanied by the introduction of subtle mutations (3), gene replacement (4) and the deletion of large genomic regions to inactivate genes or geneclusters (5). However, Cre-mediated gene manipulation requires transient transfection ofthe mutant, loxP-containing ES cells witha vectorencoding Cre recombinase and an additional round of selection toscreen for ES cell clones carrying the desired deletion. This additional manipulation of the cells in culture is unpleasing since it might affect the capacity of ES cells to contribute to the generation of germ cells in chimeric mice, which is a serious problem in gene targeting technology. The Cre-loxP recombination system has also been used to manipulate genes in a cell type-specific (6-8) or inducible (9) manner. If two loxP sites are introduced into the genome such that they are flanking an essential part of a target gene without affecting its function, Cre-mediated deletion of the loxP-flanked segment will lead to the inactivation of the gene. Gene inactivation in vivo can be restricted to a particular cell type or time point by crossing mice with a loxP-flanked target gene to transgenic mouse strains expressing cre under the control of a cell type-specific or inducible promoter. Such experiments often involve the generation of two independent mouse strains, one harbouring a loxP-flanked gene, suitable for conditional inactivation, and the other with a deletion of the respective gene to study the gene's function during mouse development. However, this requires the injection of different mutant ES cell clones into blastocysts to generate two independent mouse strains, which is expensive and time consuming.These problems of Cre-loxP mediated gene targeting can be largely overcome with the help of a cnr-transgenic mouse strain which we have generated. This strain mediates deletion of loxP-flanked genes in all tissues, including germ cells (Fig. 1). The strain was produced by injection of a DNA fragment