Reference - Detail
| RRC ID | 83747 |
|---|---|
| Author | Bussmann J, Schulte-Merker S. |
| Title | Rapid BAC selection for tol2-mediated transgenesis in zebrafish. |
| Journal | Development |
| Abstract |
The generation of zebrafish transgenic lines that express specific fluorophores in a cell- or tissue-specific manner is an important technique that takes full advantage of the optical clarity of the embryo. Identifying promoter fragments that faithfully recapitulate endogenous expression patterns and levels is often difficult and using large genomic DNA fragments, such as bacterial artificial chromosomes (BACs), makes the process of transgenesis less reliable. Here we provide a detailed protocol that allows for BAC selection and subsequent rapid modification through recombineering in Escherichia coli, resulting in BACs that can be injected into zebrafish embryos and, aided by tol2-mediated transgenesis, reliably yield stable transgenic lines. A number of BACs can be prepared in parallel, and injection of the BACs containing CFP/YFP/RFP or Gal4 cassettes allows for immediate testing of whether a particular BAC will yield the desired result. Furthermore, since injected embryos often show widespread expression, recombineered BACs provide an alternative to two-color in situ hybridizations: BACs injected into embryos of a different transgenic reporter line thus enable in vivo colocalization studies. Using this protocol, we have generated 66 stable lines for 23 different genes, with an average transgenesis rate above 10%. Importantly, we provide evidence that BAC size shows no apparent correlation to the transgenesis rate achieved and that there are no severe position effects. |
| Volume | 138(19) |
| Pages | 4327-32 |
| Published | 2011-10-1 |
| DOI | 10.1242/dev.068080 |
| PII | dev.068080 |
| PMID | 21865323 |
| MeSH | Animals Bacterial Proteins / metabolism Chromosomes, Artificial, Bacterial / genetics* DNA Primers / genetics Escherichia coli / metabolism Genetic Techniques* Genetic Vectors Green Fluorescent Proteins / metabolism In Situ Hybridization Luminescent Proteins / metabolism Polymerase Chain Reaction RNA, Messenger / metabolism* Recombination, Genetic Red Fluorescent Protein Transgenes Transposases / metabolism* Zebrafish / genetics* |
| IF | 5.611 |
| Altmetric score |
オルトメトリクス指標項目
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| The most frequently cited source | Patent(IFI CLAIMS) |
| Total number of mentions | 5 |
| Altmetric score changes over past 6months | 0.0 |
| Resource | |
| Zebrafish | UAS:GFP |