TARGETED CORRECTION OF A MAJOR HISTOCOMPATIBILITY CLASS-II E-ALPHA-GENE BY DNA MICROINJECTED INTO MOUSE EGGS
TARGETED CORRECTION OF A MAJOR HISTOCOMPATIBILITY CLASS-II E-ALPHA-GENE BY DNA MICROINJECTED INTO MOUSE EGGS
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DOI：
10.1073/pnas.86.18.7087
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发表时间：
1989-09-01
影响因子：
11.1
通讯作者：
PALMITER, RD
中科院分区：
文献类型：
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作者：
BRINSTER, RL;BRAUN, RE;PALMITER, RD
含有功能性主要组织相容性II类E α的5“末端的DNA分子。基因注射到携带E α的小鼠卵中。包含启动子和第一外显子的630个碱基对（bp）缺失的基因。通过在约500只掺入注射DNA的转基因小鼠中的1只中进行同源重组来校正缺失。校正的E α基因传递给后代，后代被培育成纯合性。Southern印迹分析、跨越缺失的DNA的聚合酶链反应扩增和序列分析揭示，校正的等位基因类似于野生型E α。基因在单碱基对多态性位点，显然是随机转换为供体或受体序列。此外，还引入了许多点突变。从校正的等位基因以组织特异性方式产生mRNA，但它们的大小不同于野生型等位基因，并且它们不产生可检测的E α。蛋白该实验证明了将外源DNA靶向在受精小鼠卵中完全失活的基因的可行性。
DNA molecules containing the 5'' end of a functional major histocompatibility class II E.alpha. gene were injected into mouse eggs bearing E.alpha. genes with 630-base-pair (bp) deletions encompassing the promoter and first exon. The deletion was corrected by homologous recombination in 1 of about 500 transgenic mice that incorporated the injected DNA. The corrected E.alpha. gene was transmitted to progeny, which were bred to homozygosity. Southern blot analysis, polymerase chain reaction amplification of the DNA spanning the deletion, and sequence analysis revealed that the corrected allele resembles the wild-type E.alpha. gene. At sites of single-base-pair polymorphisms, there was apparently random conversion to either the donor or recipient sequence. In addition, many point mutations were introduced. mRNAs were produced from the corrected allele in a tissue-specific manner, but their sizes were different from the wild-type allele, and they did not produce detectable E.alpha. protein. This experiment demonstrates the feasibility of targeting foreign DNA to a gene that is completely inactive in fertilized mouse eggs.