Regulated gene expression in transfected primary chicken erythrocytes.
We describe a method for studying transient gene expression in primary avian erythroid cells that involves controlled osmotic shock, followed by DNA transfection using DEAE-dextran. Cells treated in this way reproducibly express high levels of chloramphenicol acetyltransferase (CAT) when transfected with a plasmid having the cat gene coupled to an appropriate viral promoter. An observed correlation between levels of CAT expression and extent of hemoglobin release during controlled shock makes it possible to choose optimum conditions for expression in erythroid cells at various stages of embryonic development. Using these techniques, we have investigated the effect on CAT expression of fusing to the cat gene various portions of the chicken adult beta-globin (beta A) gene. We show that in 9-day or 12-day embryonic erythrocytes, the promoter activity of the 5' flanking region of the beta A gene (in the absence of any viral promoters) is strongly stimulated by a downstream sequence, located in the region 110-588 base pairs on the 3' side of the poly(A) signal, that acts as an enhancer. Its activity is reduced in 5-day embryonic cells and absent in primary chicken fibroblasts and mouse L cells, suggesting that this transient expression system will be useful in studying developmentally regulated globin gene expression.
Hesse
J E
JE
Nickol
J M
JM
Lieber
M R
MR
Felsenfeld
G
G
D000123:Acetyltransferases / Q000235:genetics; D000367:Age Factors; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D002463:Cell Membrane Permeability; D015500:Chloramphenicol O-Acetyltransferase; D003001:Cloning, Molecular; D004742:Enhancer Elements, Genetic; D004912:Erythrocytes / Q000502:physiology*; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics*; D014162:Transfection
D000643:Ammonium Chloride; D005914:Globins; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase