title: Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos.
abstract: The formation of transgenic chimeric chickens for use in developmental studies and as intermediates in the production of transgenic chickens requires the incorporation of stably transfected blastodermal cells into a chimera. To obtain blastodermal cells, area pellucidae of stage X (Eyal-Giladi and Kochav, Dev. Biol. 49:321-337, 1976:E.-G.&K.) embryos were collected from unincubated, freshly oviposited Barred Plymouth Rock eggs and dissociated in 0.25% trypsin/0.04% EDTA (w/v) and 2% (v/v) chicken serum in phosphate-buffered saline (Ca2+ and Mg2+ free) at 4 degrees C for 10 min. The blastodermal cells were suspended in Dulbecco's Modified Eagle's Medium (DMEM) and transfected by lipofection with superhelical pmiwZ, a plasmid containing a hybrid lacZ gene encoding bacterial beta-galactosidase (beta-gal) under the control of a chicken beta-actin/Rous sarcoma virus promoter. A mixture of 2.5 micrograms Lipofectin and 1.56 micrograms pmiwZ in 250 microliters DMEM was incubated for 30 min at 37 degrees C and added to 500 microliters of 20-40,000 cells in suspension. Cells incubated with the transfection reagents in the presence or absence of pmiwZ were either plated and cultured for 48 h at 37 degrees C in 5% CO2/95% air, or injected through a shell window into the subgerminal cavity of White Leghorn stage X (E.-G.&K.) embryos previously exposed to 500-600 rads from a 60Co source, after which the window was sealed and the egg incubated at 38 +/- 1 degrees C for 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Animal and Poultry Science, University of Guelph, Ontario, Canada.
authors.forename: R A
authors.initials: RA
authors.lastname: Fraser
authors.forename: R S
authors.initials: RS
authors.lastname: Carsience
authors.forename: M E
authors.initials: ME
authors.lastname: Clark
authors.forename: R J
authors.initials: RJ
authors.lastname: Etches
authors.forename: A M
authors.initials: AM
authors.lastname: Gibbins
mesh_terms: D000818:Animals; D001756:Blastoderm / Q000166:cytology*; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens / Q000235:genetics*; D011828:Radiation Chimera / Q000235:genetics*; D014162:Transfection / Q000235:genetics / Q000379:methods*