Regulated gene expression in the chicken embryo by using replication-competent retroviral vectors

Abstract

Rous sarcoma virus (RSV)-derived retroviral vector could efficiently deliver the green fluorescent protein (GFP), which is driven by the internal cytomegalovirus enhancer/promoter, into restricted cell populations in the chicken embryo. RSV-derived vectors coupled with the tet regulatory elements also revealed doxycycline-dependent inducible GFP expression in the chicken embryo in ovo.

FIG. 1.

Retroviral vectors. RCASBP(A)EGFP and RCASBP (B)EGFP [RCASBP(A/B)EGFP] are replication-competent retroviral vectors carrying a GFP reporter gene. GFP is produced from one of the alternative splice variants for the viral mRNA that is transcribed under control of the viral promoter within the long terminal repeat (LTR). RCANBP retroviral vectors lack a splice acceptor (SA) sequence downstream of the env region. Accordingly, expression of a transgene depends on the activity of an internal promoter. RCANBP(A/B)CMV/EGFP carries the CMV promoter and a GFP reporter gene. RCANBP(A/B)CMV/rtTA carries the CMV promoter and the rtTA. RCANBP(A/B)TRE/EGFP carries the TRE promoter and a GFP reporter gene. gag, pol, and env denote the location of viral genes. SD, splice donor.

FIG. 2.

GFP expression driven by the internal CMV promoter on E8.5 embryos. Embryos and cryosections are shown after infection with either RCASBP(B)EGFP (A, B, E, F, I, J, M, N, Q, and R) or RCANBP(B)CMV/EGFP (C, D, G, H, K, L, O, P, S, and T). Heads (A to D) and bodies (E to H) are shown with an epifluorescence dissecting microscope. Arrows (H) indicate proliferating zones in developing bones. (E to H) Ht, heart; Lv, liver. (I to L) Horizontal sections of the eye. Arrows (I, K, and L) indicate the pigmented epithelium. Arrowheads (J) indicate the neural retina. Le, lens; NR, neural retina; ON, optic nerve; PE, pigmented epithelium. Bars, 1 mm. (M to P) Comparison of the expression patterns of GFP (N and P) and the viral gag protein p19 (M and O) in the spinal cord. Arrowheads (P) indicate the dorsal root and the dorsomedial region of the DRG. DG, DRG; SC, spinal cord; V, vertebra. Bars, 200 μm. (Q to T) Comparison of the expression patterns of GFP (R and T) and p19 (Q and S) in the liver. Bars, 100 μm.

FIG. 3.

DOX-induced GFP expression in the chicken embryo. The eyes of embryos without DOX (A to D) or with DOX (E to H) are shown following infection with both RCANBP(B)CMV/rtTA and RCANBP(A)TRE/EGFP. (A and E) GFP expression in eyes. Arrows (E) indicate the pigmented epithelium. Dotted boxes are shown in the following panels (B to D and F to H) with higher magnification. Le, lens; ON, optic nerve; PE, pigmented epithelium. Bars, 1 mm. Retinas are shown with DAPI (4",6"-diamidino-2-phenylindole) staining (B and F), immunostaining against p19 (C and G), and GFP fluorescence (D and H). GC, ganglion cell layer; NR, neural retina; PE, pigmented epithelium. Bars, 50 μm. Embryos treated without (I and J) or with (L and M) DOX are shown following infection with both RCANBP(B)CMV/rtTA and RCANBP(A)TRE/EGFP. GFP expression is also shown in cryosections of the liver from embryos without (K) and with (N) DOX. Panels J and K represent the highest background expression of GFP without DOX. Bars, 100 μm. (O) Immunoblot analysis of liver lysates for GFP. Liver lysates were prepared from embryos infected without virus (lane a), with RCASBP(B)EGFP (lane b), with RCANBP(B)CMV/EGFP (lane c), and with both RCANBP(B)CMV/rtTA and RCANBP(A)TRE/EGFP (lanes d to k). Embryos without (lanes a to g) or with (lanes h to k) DOX are shown. The 2.7-kDa protein bands correspond to GFP. (P) Relative fluorescence intensity for GFP. Four samples in each experimental group were measured, and the averages are shown.