In ovo delivery of DNA to the avian embryo.
We have developed a simple method of transfecting avian embryos in ovo with various plasmid vectors that results in protein expression in the embryo. Using the chloramphenicol acetyl transferease (CAT) reporter gene, we were able to show that transfecting avian embryos with a plasmid/neutral lipid/dimethylsulfoxide (DMSO) mixture delivered to the air cell, is better than transfecting naked DNA or cationic lipid encapsulated DNA, using DMSO (P < 0.05). This method resulted in CAT expression in several avian embryonic tissues of all the embryos inoculated. We found that both the cytomegalovirus (CMV) and chicken beta actin promoters worked significantly better (P < 0.05) than the Rous sarcoma virus promoter in vitro for reporter gene expression after cationic liposome-mediated transfection. However, after in ovo delivery with neutral lipid encapsulation and DMSO mediated delivery, no significant difference (P > 0.05) between the various promoters could be determined. We believe this neutral lipid encapsulation method may represent an important platform for delivery of DNA to the avian embryo.
Oshop
Gretchen L
GL
VA-MD Regional College of Veterinary Medicine, University of Maryland, 8075 Greenmead Dr, College Park, MD 20742-3711, USA.
Elankumaran
S
S
Vakharia
Vikram N
VN
Heckert
Robert A
RA
D000199:Actins / Q000235:genetics; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002460:Cell Line / Q000378:metabolism; D002642:Chick Embryo / Q000276:immunology*; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D004121:Dimethyl Sulfoxide; D004306:Dose-Response Relationship, Immunologic; D004337:Drug Carriers; D004531:Eggs; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000008:administration & dosage*; D015707:HN Protein / Q000235:genetics / Q000276:immunology; D007243:Infectious bursal disease virus / Q000235:genetics / Q000276:immunology*; D008055:Lipids; D008081:Liposomes; D008099:Liver / Q000276:immunology; D018482:Muscle, Skeletal / Q000276:immunology; D009522:Newcastle disease virus / Q000235:genetics / Q000276:immunology*; D011201:Poultry Diseases / Q000517:prevention & control*; D011401:Promoter Regions, Genetic; D013047:Specific Pathogen-Free Organisms; D014162:Transfection; D014611:Vaccination / Q000379:methods / Q000662:veterinary*; D019444:Vaccines, DNA / Q000276:immunology*; D015678:Viral Structural Proteins / Q000235:genetics / Q000276:immunology; D014765:Viral Vaccines / Q000276:immunology*
D000199:Actins; D000956:Antigens, Viral; D004337:Drug Carriers; D015707:HN Protein; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; C082513:VP2 protein, infectious bursal disease virus; C478244:VP3 protein, infectious bursal disease virus; C478245:VP4 protein, infectious bursal disease virus; D019444:Vaccines, DNA; D015678:Viral Structural Proteins; D014765:Viral Vaccines; D015500:Chloramphenicol O-Acetyltransferase; D004121:Dimethyl Sulfoxide