A Plasmid Set for Efficient Bacterial Artificial Chromosome (BAC) Transgenesis in Zebrafish

Abstract

Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs. Using BACs containing these targeting cassettes, we show that transgenesis is as efficient as iTol2, that preselecting for expression of the transgenesis marker increases the transgenesis rate, and that BAC transgenics faithfully recapitulate the endogenous gene expression patterns and allow for the estimation of the endogenous gene expression levels.

Keywords: BAC transgenesis; gene expression; zebrafish.

Figure 1

Transgenic constructs and tol2-mediated transgenesis rates for BACs. (A) Schematic representation of BAC backbone targeting cassettes containing arms of homology for the BAC backbone (orange), a selection marker (green) flanked by Tol2 sites (purple), followed by the cryaa promoter (gray) driving a fluorescent protein (rainbow) in the lens of the eye (see Table 1). (B) Schematic representation of the BAC transgenes. Note that cxcr4b-GFP-IRES-Kate2-CaaX is not shown. (C) Rates of transgenesis for the BAC transgenes depicted in B. The size of the transgenes and the number of fish screened (n) are indicated. (D) Transgenesis rate of embryos injected with the sdf1a:sdf1a-Flag₃-HA₄ transgene, and presorted for mosaic fluorescent protein expression in the lens compared to injected embryos without mosaic fluorescent protein expression in the lens. The overall rate of transgenesis for the sdf1a:sdf1a-Flag₃-HA₄ transgene is indicated in C. (E) Rate of germline mosaicism in the transgenic founder fish for the transgenes indicated in B. Dots represent the rate of germline mosaicism in individual fish and the horizontal line indicates the median.

Figure 2

BAC transgenes faithfully recapitulate endogenous expression patterns. Overview images of embryos expressing sdf1a:sdf1a-GFP. (A) Stained embryo at 30 hours postfertilization (hpf), (B) cxcr4b:cxcr4b-Kate2-IRES-GFP-CaaX (live 36-hpf embryo), and (C) cxcr4b:Lifeact-Citrine (live 36-hpf embryo). Images are maximum (A) or sum intensity (B and C) projections of z-stacks. Scale bar corresponds to 300 μm.

Figure 3

Integration site effects on BAC transgene expression levels. Quantification of the average citrine (A) and Kate2 (B) fluorescence within the posterior lateral line primordium at 36 hpf for the cxcr4b:Lifeact-Citrine and cxcr4b:cxcr4b-GFP-IRES-Kate2-CaaX transgenes in different transgenic lines. Expression levels vary about three- to four-fold among the different transgenic lines for both BAC transgenes, likely reflecting effects of the integration site on expression levels. Individual measurements of the average fluorescence intensity within the posterior lateral line primordium are indicated by gray dots. Horizontal lines indicate the average fluorescence intensity of the different transgenic lines, and error bars indicate the 95% confidence interval.