Transposon-mediated BAC transgenesis in zebrafish and mice

Abstract

Background: Bacterial artificial chromosomes (BACs) are among the most widely used tools for studies of gene regulation and function in model vertebrates, yet methods for predictable delivery of BAC transgenes to the genome are currently limited. This is because BAC transgenes are usually microinjected as naked DNA into fertilized eggs and are known to integrate as multi-copy concatamers in the genome. Although conventional methods for BAC transgenesis have been very fruitful, complementary methods for generating single copy BAC integrations would be desirable for many applications.

Results: We took advantage of the precise cut-and-paste behavior of a natural transposon, Tol2, to develop a new method for BAC transgenesis. In this new method, the minimal sequences of the Tol2 transposon were used to deliver precisely single copies of a approximately 70 kb BAC transgene to the zebrafish and mouse genomes. We mapped the BAC insertion sites in the genome by standard PCR methods and confirmed transposase-mediated integrations.

Conclusion: The Tol2 transposon has a surprisingly large cargo capacity that can be harnessed for BAC transgenesis. The precise delivery of single-copy BAC transgenes by Tol2 represents a useful complement to conventional BAC transgenesis, and could aid greatly in the production of transgenic fish and mice for genomics projects, especially those in which single-copy integrations are desired.

Figure 1

A scheme for Tol2-mediated BAC transgenesis. (a) Structures of Tol2 and iTol2 cassette. Tol2 encodes a single transposase mRNA (solid and dotted lines). The cis-sequences required for transposition, 200 bp from the left (L200) and 150 bp from the right (R150) are shown by red triangles. In the iTol2 cassette, these are inverted outwards. (b) The cassette was inserted into a Fugu evx1 BAC tagged with Gal4FF (purple box) that included part of the hoxAa cluster. The gray box indicates the pBeloBAC11 vector. The Tol2-BAC was co-injected with the transposase mRNA into zebrafish or mouse eggs. The Tol2-BAC portion was excised in vivo and integrates as a single copy into a single genomic locus (asterisk). (c) 50 ng/μl Tol2-BAC and 25 ng/μl transposase mRNA were co-injected into one-cell stage zebrafish embryos, or 14.5 ng/μl DNA and 10 ng/μl transposase mRNA into mouse oocytes (B6C3F1). A single needle was used to inject the DNA/RNA mixture into either pronucleus, cytoplasm or both of mouse oocytes.

Figure 2

Stable Tol2-BAC transgenesis in zebrafish and mice. (a, b) Side views of double transgenic larvae (F1-18 and F1-43) carrying Tol2-Frevx1:Gal4-BAC (Tol2-BAC) and UAS:GFP that expressed GFP in spinal cord neurons at 2 dpf. (c) The insertion in F1-18 was located ~50 kb upstream of cng3. (d) The insertion in F1-43 was located in intron 7 of ntt4. 8 bp target site duplications are shown. (e) The structure of Tol2-BAC. Tol2 cis-sequences are shown by red triangles. Black bars indicate exons of hoxa and evx1 genes. A purple box indicates Gal4FF. The pBeloBAC11 vector is shown in gray and a black arrow within it is chloramphenicol. Positions of PCR fragments amplified (g, j) are shown as red lines with numbers. Positions of the BamHI, NheI and EcoRV sites used for Southern blot are shown. (f) Southern blot analysis of transgenic fish (F1-18 and F1-43) by using a ³²P-labeled Gal4FF probe. Single bands (arrowheads) were detected in DNA digested with EcoRV (~19-22 kb) and NheI (~21 kb). (g) Electrophoresis of PCR products amplified from transgenic fish. (h) PCR genotyping of F1 progeny from No.1 and No.3 founders mice with Gal4FF primers. (i) Southern blot analysis of F1 transgenic mice by using a ³²P-labeled Gal4FF (left and middle panels) or Chloramphenicol (rightmost panel) probe. Single bands (arrowheads) were detected in DNA digested with EcoRV (~23 kb) or BamHI (~20 kb). (j) Electrophoresis of PCR products amplified from transgenic mice. M, 1 kb ladder. (k) The F1-3A insertion was located near Pomc. (l) The F1-3B insertion was located in AAHY01196472.1.