The primary focus of the lab is to develop tools for studying gene expression on a genome wide scale. We have developed a technique using high-throughput sequencing for rapidly mapping the integration sites of retroviruses and transposable elements and in collaboration with Dr. Shuo Lin at UCLA, we finished the second phase of a process to map thousands of proviral integrations in the zebrafish germline to create an archived zebrafish mutant resource. We injected high-titer pseudotyped MMLV retrovirus preparations into early zebrafish embryos (approximately the 1,000 cell stage) and raised the infected fish. Those """"""""founder"""""""" fish were outcrossed to wild-type fish and The F1 generation males were raised to sexual maturity. The males were then sacrificed and the sperm cryopreserved, with a matching tissue biopsy being collected for each fish. The genomic DNA was purified and used for PCR amplification of the genomic regions directly adjacent to the proviral integration site using linker-mediated PCR. Using an indexing technique we developed, 500 PCR samples were sequenced together using the HiSeq2000 platform. Genomic locations were determined using high-throughput sequence mapping Bowtie software and sample assignment extracted from the index. We mapped >17,000 retroviral integrations and demonstrated the efficacy of our mutagenesis strategy. We have generated >3,700 predicted mutations. We recently expanded our mapping efforts through collaborations to map other insertional mutagens such as the transposable elements TOL2 and AC/DS. We have maped >3,000 of these integrations for our collaborators and will add another 1000 genes in the next two years, adding even more mutations to the zebrafish research community. We have or are working with with Dr. Stephen Ekker, Dr. Karuna Sampath, and Dr. Harold Burgess with TOL2 and AC/DS gene traps. We have recently shifted our mutagenesis efforts to using the bacterial CRISPR/Cas9 technique. This provides us with a cost effective way to efficiently target gene KO's in zebrafish. In the past year we have targeted over 165 genomic locations and identified the mutations transmitted through the germline. We will design efficient, high-throughput techniques to generate approximately 2,000 targeted gene KO's in the next 3 years. These mutations will feed into other ongoing projects in the lab.
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