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Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions.

ABSTRACT: Using a combination of techniques we developed, we infected zebrafish embryos using pseudotyped retroviruses and mapped the genomic locations of the proviral integrations in the F(1) offspring of the infected fish. From F(1) 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 approximately 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(1) 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(1) fish, it is now possible to create a stable resource that contains mutations in every known zebrafish gene.

SUBMITTER: Wang D

PROVIDER: S-EPMC1924792 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UNLABELLED:One lineage of human endogenous retroviruses (HERVs), HERV-K(HML2), is upregulated in many cancers, some autoimmune/inflammatory diseases, and HIV-infected cells. Despite 3 decades of research, it is not known if these viruses play a causal role in disease, and there has been recent interest in whether they can be used as immunotherapy targets. Resolution of both these questions will be helped by an ability to distinguish between the effects of different integrated copies of the virus (loci). Research so far has concentrated on the 20 or so recently integrated loci that, with one exception, are in the human reference genome sequence. However, this viral lineage has been copying in the human population within the last million years, so some loci will inevitably be present in the human population but absent from the reference sequence. We therefore performed the first detailed search for such loci by mining whole-genome sequences generated by next-generation sequencing. We found a total of 17 loci, and the frequency of their presence ranged from only 2 of the 358 individuals examined to over 95% of them. On average, each individual had six loci that are not in the human reference genome sequence. Comparing the number of loci that we found to an expectation derived from a neutral population genetic model suggests that the lineage was copying until at least ?250,000 years ago. IMPORTANCE:About 5% of the human genome sequence is composed of the remains of retroviruses that over millions of years have integrated into the chromosomes of egg and/or sperm precursor cells. There are indications that protein expression of these viruses is higher in some diseases, and we need to know (i) whether these viruses have a role in causing disease and (ii) whether they can be used as immunotherapy targets in some of them. Answering both questions requires a better understanding of how individuals differ in the viruses that they carry. We carried out the first careful search for new viruses in some of the many human genome sequences that are now available thanks to advances in sequencing technology. We also compared the number that we found to a theoretical expectation to see if it is likely that these viruses are still replicating in the human population today.

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Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions.

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