The mechanism of osmotic transfection of avian embryonic erythrocytes: analysis of a system for studying developmental gene expression.
We have undertaken a study of the mechanism of DNA transfer into primary chicken erythrocytes by a method named osmotic transfection. The cells are subjected to controlled osmotic swelling in NH4Cl and then ruptured in a lower osmotic strength solution containing DNA and DEAE-dextran. The osmotic rupture results in transient formation of a single hole in the cell membrane, which is followed within hours by recovery of near normal levels of RNA and protein synthesis. The association of DNA with the cells is much greater for ruptured than for unruptured cells or for cells that have been lysed and resealed before DNA is added. Transient formation of pores in the cell membrane is apparently essential for high rates of macromolecular transfer into the cell. DEAE-dextran increases the amount of DNA associated with the cells, especially after cell rupture. Our understanding of the mechanism has allowed us to extend the application of osmotic transfection to essentially all developmental stages of avian erythroid differentiation. Osmotic transfections were done with plasmids containing the chloramphenicol acetyl transferase (cat) gene placed between the chicken beta-globin promoter and the 3' beta-globin enhancer. The pattern of CAT expression at sequential developmental stages parallels that of the endogenous gene, showing that osmotically transfected cells appear to retain developmental fidelity. The approach provides a convenient, sensitive, and flexible system for the study of transient gene expression as a function of development.
D000123:Acetyltransferases / Q000097:blood / Q000235:genetics; D000818:Animals; D002642:Chick Embryo; D015500:Chloramphenicol O-Acetyltransferase; D004742:Enhancer Elements, Genetic; D004910:Erythrocyte Membrane / Q000648:ultrastructure; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D005796:Genes; D050437:Genes, Developmental; D005914:Globins / Q000235:genetics*; D006454:Hemoglobins / Q000032:analysis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D014158:Transcription, Genetic
D006454:Hemoglobins; D005914:Globins; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase