title: Developing a puncture-free in ovo chicken transfection strategy based on bypassing albumen nucleases.
abstract: Chicken is a dual-purpose animal important from both agricultural and medical aspects. Even though significant improvements have been made in chicken transgenesis technologies, chicken genome manipulation has not been widely used in developmental biology. This study was aimed to evaluate chicken egg white nuclease properties and thereof plausibility of devising an in vivo transfection technology without causing physical damage to the embryo. First, the nuclease activity of egg albumen was assessed. The egg white nucleases were strongly active in degrading DNA and RNA. The egg white DNase activity was comparable to commercially available DNase-I. Nuclease activities were also assessed after heating, proteinase K, or EDTA treatment. Unlike proteinase K, both heating and EDTA were noticeably effective for the nuclease inactivation. Simultaneous application of lipoplex form of DNA (1 μg pDB2: 3 μl Lipofectamine2000) and EDTA showed a synergistic effect in protection against egg white nucleases. Finally, we injected the lipoplexes with or without EDTA close to the embryo at day0, but outside the embryonic epiblast. Implementation of a scrutinized PCR assay indicated that transfection took place only when EDTA was complemented to the lipoplexes. The transfection rate of day4 embryos and the hatched chicks were 54.5 and 30.0%, respectively. EGFP expression was detected in two out of three transgenic chicks. In conclusion, this study provided a detail analysis of chicken egg albumen nuclease properties and suggested the feasibility of developing a puncture-free handmade technology for transfection of the chicken embryo.
authors.affiliation: Transgenesis Center of Excellence, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran; Department of Animal Science, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
authors.forename: Hamid-Reza
authors.initials: HR
authors.lastname: Amini
authors.affiliation: Department of Animal Science, Isfahan University of Technology, Isfahan, Iran.
authors.forename: Abbas
authors.initials: A
authors.lastname: Pakdel
authors.affiliation: Department of Animal Science, University College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.
authors.forename: Hossein Moradi
authors.initials: HM
authors.lastname: Shahr-Babak
authors.affiliation: Transgenesis Center of Excellence, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran; Department of Animal Science, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran. Electronic address: shahin.eghbal@khuisf.ac.ir.
authors.forename: Shahin
authors.initials: S
authors.lastname: Eghbalsaied
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002642:Chick Embryo; D002645:Chickens / Q000235:genetics*; D003851:Deoxyribonucleases / Q000737:chemistry; D004492:Edetic Acid / Q000494:pharmacology; D005818:Genetic Engineering / Q000379:methods / Q000662:veterinary*; D008055:Lipids / Q000494:pharmacology; D010047:Ovalbumin / Q000737:chemistry; D014162:Transfection / Q000379:methods / Q000662:veterinary*
chemical_list: D008055:Lipids; C086724:Lipofectamine; D010047:Ovalbumin; D004492:Edetic Acid; D003851:Deoxyribonucleases
keywords: Chicken; Egg albumen; Handmade; In ovo; Nuclease; Transgenesis