Searching for genes involved in the pathogenesis of primary immunodeficiency diseases: lessons from mouse knockouts.
Searching for genes involved in the pathogenesis of primary immunodeficiency diseases: lessons from mouse knockouts.
复制标题
寻找与原发性免疫缺陷疾病发病机制有关的基因：小鼠基因敲除的教训。
DOI：
10.1023/a:1027322314256
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发表时间：
1997
影响因子：
9.1
通讯作者：
Pandolfi,F
中科院分区：
文献类型：
--
作者：
Kokron,CM;Bonilla,FA;Oettgen,HC;Ramesh,N;Geha,RS;Pandolfi,F
Gene targeting by homologous recombination is a powerful technique for the development of mouse strains with desired genetic alterations. This method enables the characterization of the in vivo functions of the targeted gene. The approach has been most often used to generate null mutations, thus, genes are" knocked out," and mice derived in this way are often called" knockout mice." The knockout process arose from methods of in vitro propagation of pluripotent embryonic stem (ES) cells and the demonstration that a relatively small (10 to 20-kb) piece of transfected DNA can recombine with its homologous chromosomal counterpart (1). Vectors are constructed containing a positive selectable marker (eg, neomycin resistance) interposed between two stretches of DNA which normally flank the region to be disrupted (Fig. 1). Homologous recombination of this vector leads to replacement of the region of interest with the marker on one chromosome, that is, the ES cell is heterozygous for the mutated gene. Gene-targeted ES cells can then be injected into blastocysts followed by implantation in the uterus of a pseudopregnant female. If gene targeted ES cells have retained their pluripotency and give rise to gametes (germline) in the resultant chimeras, the gene disruption may easily be bred to homozygosity. One may then analyze the phenotype resulting from congenital absence of the disrupted genetic region (reviewed in Refs. 2-4).Several variations of this knockout strategy have been developed. The recombinase activating gene (RAG)-deficient blastocyst complementation assay is used to study the function of genes in T and B cells. Knockout mice deficient in either the RAG-1 or the RAG-2 gene lack mature T and B lymphocytes due to their inability to rearrange the gene segments which constitute the antigen receptors (5, 6). Injection of homozygous disrupted ES cells into blastocysts from RAG-1-or RAG-2-deficient mice yields chimeras in which the injected ES cells constitute the only potential origin of mature T or B lymphocytes (7). ES cells homozygous for disrupted genes may be generated either by sequential transfections with constructs bearing different selectable markers (8) or by culture of ES cells under stringent conditions which permit the selection of cells homozygous for the disruption (9). This is a convenient approach for the study of mutations which would be lethal if expressed in all cell lineages of the mouse. Other modifications of the knockout technique permitting even more subtle genetic manipulations have been described. The CrelLoxP strategy provides another way to study the role of a particular gene product in only one or a few types of cells. In the CrelloxP method (10), the
