Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions
Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions
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DOI：
10.1073/pnas.0705502104
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发表时间：
2007-07-24
影响因子：
11.1
通讯作者：
Burgess, Shawn M.
中科院分区：
文献类型：
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作者：
Wang, Dongmei;Jao, Li-En;Burgess, Shawn M.
使用我们开发的技术组合，我们使用伪型逆转录病毒感染斑马鱼胚胎，并绘制了受感染鱼后代F中原病毒整合的基因组位置。从F， fish，我们获得了2,045个序列，代表933个独特的逆转录病毒整合。共有599个可与当前的基因组组装（Zv6）相匹配，其中233个整合在基因内。通过近亲繁殖携带25种不同基因的原病毒整合的鱼，我们能够证明，在大约50%的基因“命中”中，mRNA转录水平降低了>= 70%，如果整合在外显子或第一个内含子中，则发生突变的可能性最高。根据这些数据，逆转录病毒的致突变频率接近五分之一。此外，当小鼠白血病病毒特异性地整合在基因的第一个内含子上而不整合在其他内含子上时，可以观察到强烈的诱变效应。19个基因失活事件中有3个有胚胎缺陷。使用我们概述的策略，可以在F，鱼上每30个测序反应中确定1个诱变事件。与目前在斑马鱼中用于识别基因突变的重测序方法（针对基因组中诱发的局部病变（TILLING））相比，这一效率提高了20到30倍。将这种效率的提高与冷冻保存来自F鱼的精子样本相结合，现在有可能创造一种稳定的资源，其中包含所有已知的斑马鱼基因突变。
Using a combination of techniques we developed, we infected zebrafish embryos using pseuclotyped retroviruses and mapped the genomic locations of the proviral integrations in the F, offspring of the infected fish. From F, fish, we obtained 2,045 sequences representing 933 unique retroviral integrations. A total of 599 were mappable to the current genomic assembly (Zv6), and 233 of the integrations landed within genes. By inbreeding fish carrying proviral integrations in 25 different genes, we were able to demonstrate that in approximate to 50% of the gene "hits," the mRNA transcript levels were reduced by >= 70%, with the highest probability for mutation occurring if the integration was in an exon or first intron. Based on these data, the mutagenic frequency for the retrovirus is nearly one in five integrations. In addition, a strong mutagenic effect is seen when murine leukemia virus integrates specifically in the first intron of genes but not in other introns. Three of 19 gene inactivation events had embryonic defects. Using the strategy we outlined, it is possible to identify 1 mutagenic event for every 30 sequencing reactions done on the F, fish. This is a 20- to 30-fold increase in efficiency when compared with the current resequencing approach [targeting induced local lesions in genomes (TILLING)] used in zebrafish for identifying mutations in genes. Combining this increase in efficiency with cryopreservation of sperm samples from the F, fish, it is now possible to create a stable resource that contains mutations in every known zebrafish gene.