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First published online 26 January 2005
doi: 10.1242/dev.01652

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Ubiquitous GFP expression in transgenic chickens using a lentiviral vector

¹ University of Utah School of Medicine, Department of Neurobiology and Anatomy, and Children's Health Research Center, Room 401 MREB, 20 North 1900 East Salt Lake City, Utah 84132-3401, USA
² University of Utah School of Medicine, Department of Pediatrics, Room 2R063, 20 North 1900 East Salt Lake City, Utah 84132-2204, USA
³ GeneWorks, Avian Transgenics, 3950 Varsity Drive, Ann Arbor, Michigan 48108, USA
⁴ University of Ghana Medical School, Accra, Ghana, West Africa
⁵ Tranzyme Pharma, PO Box 13097, 21 Davis Drive, Research Triangle Park, North Carolina 27709, USA

View larger version (104K): [in a new window]   Fig. 1. PCR transgene analysis. (A) Semen from six chimeric G0 roosters was analyzed by PCR for the presence of the transgene. Three roosters were positive for the transgene in the germline, but only rooster 6 was able to pass the transgene onto his offspring. (B) Two mature offspring from rooster 6 had their semen tested for the presence of the transgene. Increasing template quantity from one of these G1 heterozygous roosters showed the expected quantity-dependent band increase. Neg, negative control; Pos, positive control; MW, molecular weight marker.

View larger version (63K): [in a new window]   Fig. 2. GFP expression in whole-mount and sectioned transgenic embryos. (A-D) Examination by brightfield (A,C) or fluorescence (B,D) illumination of a live whole-mount 72-hour embryo (A,B) and a 72-hour embryo after sectioning and processing with anti-GFP antibody (C,D). GFP expression is ubiquitous (B,D). White line in A shows angle of the section. Scale bar: in A, 150 µm for A,B; in C, 100 µm for C,D.

View larger version (52K): [in a new window]   Fig. 3. E14 chick organs express GFP uniformly. Fresh organs from E14 embryos were dissected out and checked for GFP using fluorescence microscopy. Kidneys (A-C), eye (D-F), heart (G-I) and brain (J-L) are all positive for GFP expression. Lung, liver, pancreas, intestine and gizzard also uniformly express GFP (not shown). (C) Transverse section of the left kidney shown in A. (F) Magnified image of the right anterior portion of the transverse section of the eye in D. The heart in G was sectioned through the plane of the paper. L is an oblique section through the caudal part of the brain in J, including the tectum, cerebellum and brain stem. Lines in A,D and J indicate level of sections in C,F and L, respectively. a, apex; bs, brain stem; c, choroid; cb, cerebellum; ch, cerebral hemisphere; l, lens; ms, mesonephros; mt, metanephros; ot, outflow tract; r, retina; rpe, retinal pigment epithelium; s, sclera; t, tectum; v, ventricle. Scale bars: in A, 150 µm for A,B,D,E,G-K; in C, 50 µm; in F, 40 µm; in L, 35 µm.

View larger version (112K): [in a new window]   Fig. 4. Primitive streak graft using transgenic chick tissue. Wild-type chick host embryo at HH stage 3d grafted with a segment of the primitive streak obtained just caudal to Hensen's node from a transgenic chick donor embryo. (A) The location of the grafted tissue in the host (white arrowheads) is shown immediately after grafting. After 1 hour the graft had fully integrated. (B) The grafted embryo after 20 hours of incubation in EC culture. (C) Following immunocytochemistry with anti-GFP antibody and DAB, the grafted cells can be identified within the heart tube and as bilateral streams extending caudally into the area pellucida. The horizontal line indicates the level of the section shown in D. (D) Paraffin sections of the embryo (12 µm) show that the labeled cells (brown) contributed mainly to the heart mesoderm at the level (anterior intestinal portal) of the fusing lateral body folds. Scale bars: in A,D, 150 µm; in B, 250 µm for B,C.

View larger version (76K): [in a new window]   Fig. 5. Somite graft using transgenic chick tissue. (A) Three somites from a transgenic embryo (black arrowheads) were grafted into the left side of a 48-hour wild-type chick host embryo (ventral side up in EC culture). (B) After a 24-hour incubation, the integrated graft was observed on the left side of the whole-mount embryo (dorsal view) after processing for immunocytochemistry with an anti-GFP antibody and DAB staining. (C) Higher magnification view of the integrated graft in B. (D) Section at the level indicated by the horizontal line in B. Paraffin section (12 µm) at the level of the donor somitic tissue demonstrates that cells integrate into the host embryo in the neural tube, dermomyotome and heart. Scale bars: in B, 200 µm for A,B; in C, 200 µm for C,D.

View larger version (105K): [in a new window]   Fig. 6. In situ hybridization (ISH) and GFP expression in wholemount embryos. Chd (A,B), Ganf (C,D) and Sox2 (E,F) were used as ISH probes in whole-mount embryos. Anti-GFP antibody with an Alexa Fluor® 488-tagged secondary antibody was used to restore fluorescence following ISH (B,D,F). Scale bar: in F, 200 µm for A-F.

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