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A Shortened Life Span of EKLF-/- Adult Erythrocytes, Due to a Deficiency of  -Globin Chains, Is Ameliorated by Human  -Globin Chains
Sai-Kiang Lim,
James J. Bieker,
Chyuan-Sheng Lin, and
Frank Costantini
From the Department of Genetics and Development, Columbia University, New York, NY; and the Brookdale Center for Molecular Biology, The Mount Sinai School of Medicine, New York, NY.
Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF-/- ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF-/- colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of  h1-globin, which continued to be expressed at a very low level. The ratio of adult -globin/ -globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF-/- cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF-/- erythrocytes in adult animals must be short-lived, apparently due to the imbalance of -versus -globin chains, leading to the precipitation of excess -globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF-/- ES cells of a human LCR/ -globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.
Blood, Vol. 90 No. 3 (August 1), 1997:
pp. 1291-1299
© 1997 by The American Society of Hematology.
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