|Kagami, Hiroshi - AZABU UNIVERSITY, JAPAN|
|Matsubara, Yuko - NIAI - TSUKUBA, JAPAN|
|Naito, Mitsuru - NIAI - TSUKUBA, JAPAN|
Submitted to: Journal of Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 8, 1999
Publication Date: N/A
Interpretive Summary: No method is currently available that ensures a routine and efficient means of inserting foreign genes into poultry. Two techniques for inserting foreign DNA were investigated. The first involved the direct injection of either SV-lacZ reporter genes (transfected cells stain blue) or a RSV-GFP (transfected cells turn green when fluoresced) into freshly laid chicken eggs when the embryo contains about 60,000 cells. When SV-lacZ was injecte into the embryo, no expression of the gene was observed. If SV-lacZ was complexed with a liposome before injection, the reporter gene was expressed in a few embryos. The best result was obtained when the injected embryos were subsequently electroporated with 3 pulses of DC current at 100 volts for, 50 usec. Unfortunately, this technique was not effective for the RSV-GFP gene. The second technique involved transfection of embryonic cells in culture with RSV-GFP complexed with liposome, and then injection of the transfected cells into the embryonic disc from which about 15% of the cell had been removed. Seven of 35 surviving embryos expressed the reporter gene in circulating blood cells and vascular system 3 days after transfer. This method may be useful for producing transgenic birds because only the green fluorescing embryos need to be cultured to hatching. This procedure warrants additional testing to determine whether the transgenic birds that are hatched will contain the foreign genes in their germ cells and transmit the genes into their progeny.
Technical Abstract: Experiments were conducted to develop techniques to introduce exogenous DNA into chicken embryos at the blastula stage. Two reporter constructs for the transfection of chicken embryos were pSV-B-galactosidase (SV- Bgal) and pKK-v4-lacZ (RSV-GFP). Freshly oviposited, unincubated chicken eggs were broken to expose the embryonic disk for the DNA injection and introduction of donor cells to produce chimeric embryos. After the manipulation, the embryo and yolk was transferred into an egg shell and cultured in vitro for several days. For SV-Bgal, DNA was directly injected into the central disk area of the blastoderm. Bacterial galactosidase expression was improved by complexing DNA with a liposome before the microinjection, and further by electroporating embryos at 100 volts, 50 æsec., and 3 pulses, to achieve its expression in 75% of embryos three days later. However, the above approach was not succcessful for introducing RSV-GFP into embryos. The second approach used was to insert RSV-GFP into blastodermal cells from donor embryos by liposome-mediated transfection. Then, donor cells were introduced into recipient embryos to produce chimeras with cells carrying the reporter construct. The mortality of chimeric embryos was about 40% after three days of incubation. Electroporation of recipient embryos further increased embryo mortality. Seven out of 35 surviving embryos expressed GFP. The GFP expression was monitored, starting as early as two days after the injection. The expression of GFP was primarily in extra-embryonic bodies and cells in circulation.