Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo.
Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)
Brazolot
C L
CL
Department of Animal and Poultry Science, University of Guelph, Ontario, Canada.
Petitte
J N
JN
Etches
R J
RJ
Verrinder Gibbins
A M
AM
D000818:Animals; D001756:Blastoderm / Q000637:transplantation*; D002478:Cells, Cultured; D002642:Chick Embryo; D002678:Chimera / Q000235:genetics; D008081:Liposomes; D010714:Phosphatidylethanolamines; D010957:Plasmids; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
D008081:Liposomes; D010714:Phosphatidylethanolamines; C020888:1,2-dielaidoylphosphatidylethanolamine; D001616:beta-Galactosidase