Transposon-mediated BAC transgenesis in zebrafish

Abstract

Bacterial artificial chromosomes (BACs) are widely used in studies of vertebrate gene regulation and function because they often closely recapitulate the expression patterns of endogenous genes. Here we report a step-by-step protocol for efficient BAC transgenesis in zebrafish using the medaka Tol2 transposon. Using recombineering in Escherichia coli, we introduce the iTol2 cassette in the BAC plasmid backbone, which contains the inverted minimal cis-sequences required for Tol2 transposition, and a reporter gene to replace a target locus in the BAC. Microinjection of the Tol2-BAC and a codon-optimized transposase mRNA into fertilized eggs results in clean integrations in the genome and transmission to the germline at a rate of ∼15%. A single person can prepare a dozen constructs within 3 weeks, and obtain transgenic fish within approximately 3-4 months. Our protocol drastically reduces the labor involved in BAC transgenesis and will greatly facilitate biological and biomedical studies in model vertebrates.

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

“…BAC clone (number 74B2) from the DanioKey zebrafish BAC library containing the six2a and six3a genes was purchased from Source BioScience. Recombiant BACs were generated as previously described (36,37). For further details, see SI Materials and Methods.…”

Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena

“…To generate BAC240GFP and F3801GFP clones, the Sox3 gene region was replaced with an EGFP þ polyA cassette PCR amplified from the hsGBA-NKm plasmid 60,61 . For efficient BAC and fosmid transgenesis using the medaka Tol2 transposon, we inserted the iTol2 cassette into the vector backbone of all clones 62,63 . Each modified clone was purified using a Qiagen Large-Construct kit (Qiagen), and Tol2 transposon mRNA was synthesized in vitro using an SP6 mMessage mMachine kit (Ambion).…”

Long range gene-regulatory sequences identified by transgenic expression of bacterially-engineered enhancer-trap BACs in zebrafish

“…Recently a moderate throughput BAC modification procedure has been described that uses RedET recombination to introduce the GFP cassette and iTol2 for integration into zebrafish chromosomes [35]. Procedures for constructing transgenic animals with functionalized BACs in mice [36], or zebrafish [37], have also been reviewed recently. BAC transgenisis in zebrafish to localize distal cis-acting generegulatory sequences were highlighted recently [38].…”

“…Originally isolated from medaka fish, Tol2 is a vertebrate transposon system that functions effectively in zebrafish and mice [62]. Traditionally, Tol2 ends in the inverted orientation and flanking a 1 kb spacer DNA (iTol2) were introduced into BAC DNA using recombination of homologous sequences between targeting vector and the genomic insert [37,62]. To avoid potential complications from rearrangement of highly repetitive DNA that occurs in certain BACs from higher vertebrate genome libraries (see for example the mouse Npr3 gene locus analyzed earlier [41]), a simpler and more widely applicable approach has been developed using Tn10 transposons to deliver iTol2 ends into BACs [43].…”

“…Germline integration of BAC DNA in zebrafish was achieved using Tol2 [37,38,43]. Originally isolated from medaka fish, Tol2 is a vertebrate transposon system that functions effectively in zebrafish and mice [62].…”