Efficient production of germline transgenic chickens using lentiviral vectors

Abstract

An effective method for genetic modification of chickens has yet to be developed. An efficient technology, enabling production of transgenic birds at high frequency and with reliable expression of transgenes, will have many applications, both in basic research and in biotechnology. We investigated the efficiency with which lentiviral vectors could transduce the chicken germ line and examined the expression of introduced reporter transgenes. Ten founder cockerels transmitted the vector to between 4% and 45% of their offspring and stable transmission to the G2 generation was demonstrated. Analysis of expression of reporter gene constructs in several transgenic lines showed a conserved expression profile between individuals that was maintained after transmission through the germ line. These data demonstrate that lentiviral vectors can be used to generate transgenic lines with an efficiency in the order of 100-fold higher than any previously published method, with no detectable silencing of transgene expression between generations.

Figure 1

Schematic representation of the EIAV vectors used in this study. The grey box represents the EIAV packaging signal, and the diagonally lined box in pONY8.4GCZ represents the MLV U3 region. Restriction sites (XbaI [X], BstEII [B]) used for Southern blot analysis are indicated.

Figure 2

Southern transfer analysis of genomic DNA from individual birds to identify proviral insertions. Genomic DNA samples were digested with XbaI (A,C,D) or BstEII (B) and hybridized with a probe for lacZ. (A,B) Analysis of 14 G₁ offspring of G₀ bird no. 1-4 (Table 1) revealed multiple proviral insertions in the G₁ birds. (C) Analysis of G₁ bird no. 2-2/6 (lane 1) and nine of his G₂ offspring (lanes 2–10) and (D) G₁ bird no. 2-2/19 (lane 1) and 14 of his G₂ offspring (lanes 2–15).

Figure 3

Reporter gene expression in pONY8.0cZ and pONY8.0G G₁ transgenic birds. (A) Western blot analysis of liver (Li), heart (He), skeletal muscle (Sm), brain (Br), oviduct (Ov), skin (Sk), spleen (Sp), intestine (In), kidney (Ki), pancreas (Pa) and bone marrow (Ma) protein extracts from five adult G₁ birds each containing single, independent insertions of pONY8.0cZ. β-Galactosidase protein detected as described in Methods. (B) Sections of skin, pancreas and intestine from G₁ 2-2/19 stained for β-galactosidase activity and comparable sections of a non-transgenic control bird (arrows indicate epidermis of skin and villi of intestine). Scale bars, 0.5 mm. (C) Sections of skin, pancreas and breast muscle from a single-copy transgenic and a control bird were visualized for GFP fluorescence (arrow indicates epidermis of skin). Exposure conditions were identical for each transgenic and control pair, but differed between tissue types. Scale bars, 0.5 mm.

Figure 4

Reporter gene expression in pONY8.4GCZ G₁ transgenic birds. Sections of tissues from a single-copy G₁ bird were stained for β-galactosidase activity (arrow indicates smooth muscle of intestine). Scale bars, 0.5 mm (except final panel). In the higher magnification of the oviduct section, arrows identify cells lining the tubular gland. Scale bar, 0.05 mm.

Figure 5

Reporter gene expression in G₂ transgenic birds. (A) Western analysis of protein extracted from intestine (Int), skin (Skn), liver (Liv) and pancreas (Pan) of G₁ cockerels 2-2/19 and 2-2/6 and two G₂ offspring of each bird. (B) Top panel: five G₁ offspring of bird number 4-1. The four birds on the left are transgenic for pONY8.0G and express eGFP. The bird on the right is not transgenic. Lower panel: five G₂ offspring of bird number 4-1/66. The bird in the centre is not transgenic.