A shortened life span of EKLF-/- adult erythrocytes, due to a deficiency of beta-globin chains, is ameliorated by human gamma-globin chains.
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 beta h1-globin, which continued to be expressed at a very low level. The ratio of adult beta-globin/alpha-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 beta-versus alpha-globin chains, leading to the precipitation of excess alpha-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/gamma-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.
Lim
S K
SK
Department of Genetics and Development, Columbia University, New York, NY 10032, USA.
Bieker
J J
JJ
Lin
C S
CS
Costantini
F
F
D000818:Animals; D002454:Cell Differentiation; D002678:Chimera; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004905:Erythrocyte Aging / Q000235:genetics*; D015672:Erythroid Precursor Cells; D004920:Erythropoiesis / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting; D005812:Genes, Switch; D005813:Genes, Synthetic; D015316:Genetic Therapy; D005914:Globins / Q000172:deficiency* / Q000235:genetics*; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008099:Liver / Q000196:embryology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid; D012156:Reticulocytes / Q000378:metabolism; D013045:Species Specificity; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D017086:beta-Thalassemia / Q000097:blood* / Q000196:embryology / Q000235:genetics / Q000628:therapy
D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C080774:erythroid Kruppel-like factor; D005914:Globins