Site-specific genetic engineering of the Anopheles gambiae Y chromosome

Abstract

Despite its function in sex determination and its role in driving genome evolution, the Y chromosome remains poorly understood in most species. Y chromosomes are gene-poor, repeat-rich and largely heterochromatic and therefore represent a difficult target for genetic engineering. The Y chromosome of the human malaria vector Anopheles gambiae appears to be involved in sex determination although very little is known about both its structure and function. Here, we characterize a transgenic strain of this mosquito species, obtained by transposon-mediated integration of a transgene construct onto the Y chromosome. Using meganuclease-induced homologous repair we introduce a site-specific recombination signal onto the Y chromosome and show that the resulting docking line can be used for secondary integration. To demonstrate its utility, we study the activity of a germ-line-specific promoter when located on the Y chromosome. We also show that Y-linked fluorescent transgenes allow automated sex separation of this important vector species, providing the means to generate large single-sex populations. Our findings will aid studies of sex chromosome function and enable the development of male-exclusive genetic traits for vector control.

Keywords: SIT; biotechnology.

Fig. 1.

Site-specific genetic engineering of the Y chromosome. Overview of constructs and transgenic lines generated in this study and the stepwise approach taken to modify the Y chromosome.

Fig. 2.

Phenotype of Y-linked strains. (A) Expression of green (GFP), red (RFP), and blue (CFP) fluorescent markers in the tissues of L2 larvae shown as transmitted-light image (TM) in Left. The white arrowheads indicate GFP expression in the developing larval gonads. (B) FISH with probes designed against the X-linked rDNA (labeled with Cy5) and the pHome-T construct (labeled with Cy3) hybridized to the A. gambiae transgenic line. Mitotic chromosome slide preparations were prepared from imaginal discs of fourth instar larvae. X and Y indicate the sex chromosomes and A indicates autosomes. (C) Confocal analysis of GFP expression in dissected testes of transgenic adult males.

Fig. 3.

Flow cytometry analysis and automated sex separation of the T4 strain. (A) A total of 16,750 larvae of the T4 line were analyzed according to their level of green and red fluorescence (green and red clouds) and sorted via the gates indicated (black lines). (B) Purified larvae (6,415 individuals) gated in A as having only background fluorescence were subjected to a second sorting run in the COPAS to check for the absence of contaminating GFP positive larvae. (C) Analysis of the fluorescence profile of ∼2,500 F₁ larvae carrying the T4 transgene and expressing the I-SceI nuclease from which 1,246 GFP-positive larvae (upper compact cloud) and 902 GFP-negative larvae (lower compact cloud) were COPAS purified. (D) Analysis of approximately 9,100 F₂ larvae from an intercross of the F₁. Three classes of red fluorescent larvae are seen along the x axis, indicating normal segregation of the red-marked I-SceI transgene. Three classes of green fluorescent larvae are also seen along the y axis: high green fluorescence (6.3% of larvae), low green fluorescence (6.8% of larvae), and GFP-negative larvae (87%).