Transgenic songbirds offer an opportunity to develop a genetic model for vocal learning

Abstract

Zebra finches are widely used for studying the basic biology of vocal learning. The inability to introduce genetic modifications in these animals has substantially limited studies on the molecular biology of this behavior, however. We used an HIV-based lentivirus to produce germline transgenic zebra finches. The lentivirus encoded the GFP regulated by the human ubiquitin-C promoter [Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D (2002) Science 295:868-872], which is active in a wide variety of cells. The virus was injected into the very early embryo (blastodisc stage) to target the primordial germline cells that later give rise to sperm and eggs. A total of 265 fertile eggs were injected with virus, and 35 hatched (13%); 23 of these potential founders (F0) were bred, and three (13%) produced germline transgenic hatchlings that expressed the GFP protein (F1). Two of these three founders (F0) have produced transgenic young at a rate of 12% and the third at a rate of 6%. Furthermore, two of the F1 generation transgenics have since reproduced, one having five offspring (all GFP positive) and the other four offsping (one GFP positive).

Fig. 1.

(A) View of the zebra finch egg (≈1.5 cm long). A small hole, larger than would normally be made, has been opened to show the embryo of a freshly laid egg (arrow). (Scale bar: 1.0 mm.) (B) A higher-magnification view of an embryo showing the central region in which viral injections were concentrated (black circle). (Scale bar: 1.0 mm.)

Fig. 2.

GFP expression in different tissues of Blk5, a mosaic transgenic mouse (FO). Bright field images of tissues are shown in the left column, and the corresponding fluorescent image is presented in the right column. (A) Whole brain as seen from above, looking at the dorsal side of the brain with the caudal end at the bottom of the image. (B) Testes. (C) Muscle tissue of the inner side of the left leg.

Fig. 3.

(A) Fluorescent illumination of 2 live offspring of a transgenic founder 1 day after hatching. The undersides of the hatchlings are shown; heads are at the top. The soft down on the heads fluoresces orange-red. The hatchling on the left is negative for GFP expression, but its sibling on the right is positive. (B) PCR analysis of genomic DNA from blood. GFP primers (Lower band) test for transgene, and androgen receptor primers (Upper band) test for DNA quality. M,L = Quanti-ladder from Origene. (1) No DNA. ( and 3) Non–viral-injected adult female and male. (, , , and 8) Four siblings from the same clutch (2 negative, 2 positive). All offspring are of the same mosaic founder, which is represented in lane 6. (C) Southern blot analysis of members of 2 clutches of 5 and 3 individuals, each of which was produced by a different F0 individual (represented in lane 1). Note that each clutch has 2 GFP-positive individuals, and that their corresponding lanes show a single dark band, evidence that the transgene is present as a single copy (see Materials and Methods). The GFP-negative individuals lack this band. The absence of a detectable band in the F0 individuals presumably results from the fact that in mosaics, the total number of blood cells that carry the gene is below the detectable limit of Southern blot analysis but not that of PCR analysis.

Fig. 4.

Insertion and expression of the transgene does not inhibit song imitation. Shown are spectrograms of song from the tutor (A) and the germline transgenic male pupil (B) at age 4 months.