Rapid BAC selection for tol2-mediated transgenesis in zebrafish

TY - JOUR

T1 - Rapid BAC selection for tol2-mediated transgenesis in zebrafish

AU - Bussmann, J.

AU - Schulte-Merker, S.

N1 - Reporting year: 2011

PY - 2011

Y1 - 2011

N2 - 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. [KEYWORDS: 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, Transgenes, Transposases/ metabolism, Zebrafish/ genetics]

AB - 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. [KEYWORDS: 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, Transgenes, Transposases/ metabolism, Zebrafish/ genetics]

U2 - 10.1242/dev.068080

DO - 10.1242/dev.068080

M3 - Article

SN - 0950-1991

VL - 138

SP - 4327

EP - 4332

JO - Development

JF - Development

IS - 19

ER -