TARGET-SELECTED GENE INACTIVATION IN CAENORHABDITIS-ELEGANS BY USING A FROZEN TRANSPOSON INSERTION MUTANT BANK
TARGET-SELECTED GENE INACTIVATION IN CAENORHABDITIS-ELEGANS BY USING A FROZEN TRANSPOSON INSERTION MUTANT BANK
复制标题
DOI：
10.1073/pnas.90.16.7431
复制
发表时间：
1993-08-15
影响因子：
11.1
通讯作者：
PLASTERK, RHA
中科院分区：
文献类型：
--
作者：
ZWAAL, RR;BROEKS, A;PLASTERK, RHA
为了理解基因型如何决定动物秀丽隐杆线虫的表型，理想情况下需要知道基因组的完整序列和基因对表型的贡献，这需要有效的反向遗传学策略。我们在这里报告，Tc 1转座子诱导频繁删除的侧翼DNA，显然是由于Tc 1切除后不精确的DNA修复。我们用它分两步来复制基因。(i)我们建立了一个冷冻库的5000线虫线诱变的Tc 1插入，从插入突变体的感兴趣的基因可以恢复。它们在文库中的地址通过PCR确定。(ii)然后再次通过PCR筛选动物，以检测其中Tc 1和侧翼DNA的1000-2000个碱基对缺失的衍生物，因此目的基因失活。迄今为止，我们已经分离出Tc 1插入在16个不同的基因，并获得其中6个缺失衍生物。
To understand how genotype determines the phenotype of the animal Caenorhabditis elegans, one ideally needs to know the complete sequence of the genome and the contribution of genes to phenotype, which requires an efficient strategy for reverse genetics. We here report that the Tc1 transposon induces frequent deletions of flanking DNA, apparently resulting from Tc1 excision followed by imprecise DNA repair. We use this to inactivate genes in two steps. (i) We established a frozen library of 5000 nematode lines mutagenized by Tc1 insertion, from which insertion mutants of genes of interest can be recovered. Their address within the library is determined by PCR. (ii) Animals are then screened, again by PCR, to detect derivatives in which Tc1 and 1000-2000 base pairs of flanking DNA are deleted, and thus a gene of interest is inactivated. We have thus far isolated Tc1 insertions in 16 different genes and obtained deletion derivatives of 6 of those.