RT-PCR using RNA from EKLF+/+, EKLF+/−, and EKLF−/− ES cell-derived erythroid colonies. RNA from each erythroid colony was purified and 1/10 of the RNA was reversed transcribed and amplified by PCR using α- and β-globin specific primers to confirm that the colonies were erythroid. The rest of the RNAs were then reverse transcribed and cDNAs from three erythroid colonies of each genotype were then pooled. The resulting cDNAs were diluted 1× and 10×, and amplified by PCR in the presence of ³²P-dCTP. The PCR products were separated on a polyacrylamide gel, quantitated by phosphorimaging, and exposed to autoradiography. (a) α- and ζ-globin specific primers. Controls are embryonic yolk sac (YS) and fetal liver (FL). (b) α- and ε^y-globin specific primers. (c) α- and β-globin specific primers. (d) α- and βh1-globin specific primers. βh1-globin mRNA was not detected in the WT colonies, but was present in EKLF+/− colonies at about 1/10 the level in EKLF−/− colonies after normalizing against α-globin mRNA. ε^y-globin and ζ-globin mRNAs were not detectable in colonies of any of the three genotypes.