EXPRESSION OF HUMAN α-GLOBIN GENES IN HYBRID MOUSE ERYTHROLEUKEMIA CELLS

Abstract In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or deletions that could explain this loss of zeta-globin gene expression. These data suggest that regulation of globin gene expression in these erythroleukemia cells involves trans-acting regulatory factors specific for the adult and embryonic stages of development.

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Regulation of human fetal and adult globin genes in mouse erythroleukemia cells

Blood ◽

10.1182/blood.v78.5.1355.1355 ◽

1991 ◽

Vol 78 (5) ◽

pp. 1355-1363 ◽

Cited By ~ 18

Author(s):

BJ Morley ◽

CA Abbott ◽

WG Wood

Keyword(s):

Globin Genes ◽

Beta Globin ◽

Chromosome 11 ◽

Globin Mrna ◽

Chromosomal Organization ◽

Gamma Globin ◽

Erythroleukemia Cells ◽

Regulatory Information ◽

Beta Gene ◽

Mouse Erythroleukemia

Abstract We have examined whether transfected mouse erythroleukaemia (MEL) cells can be used to examine differential expression of human gamma- and beta- globin genes. These cells, which express only their adult globin genes, will transcribe the human adult beta gene but not the fetal gamma genes when they are introduced on an intact human chromosome 11 by cell fusion. However, MEL cells stably transfected with the human A gamma gene attached to one of the active elements (HS2) of the beta-globin locus control region (LCR) readily produce gamma-globin mRNA in amounts equivalent to those seen with a comparable beta gene insert. When both beta and gamma genes are attached to HS2, equal amounts of beta A gamma mRNAs are produced, irrespective of the gene order. Furthermore, when HS2 is inserted into the 5′ end of a 40-kb cosmid containing the G gamma A gamma-117 delta beta genes in their normal chromosomal organization (but with the Greek HPFH -117 A gamma gene mutation), it directs expression of readily detectable amounts of G gamma A gamma and beta-globin mRNAs in MEL cells. Therefore, under these circumstances we have observed no competition between beta and gamma genes for expression in MEL cells. These findings suggest that MEL cells are capable of perpetuating regulatory information involved in developmental control when it is provided by an intact chromosome, but are incapable of reconstructing such information on transfected DNA.

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Hemoglobin synthesis in somatic cell hybrids: coexpression of mouse with human or chinese hamster globin genes in interspecific somatic cell hybrids of mouse erythroleukemia cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.72.7.2682 ◽

1975 ◽

Vol 72 (7) ◽

pp. 2682-2686 ◽

Cited By ~ 18

Author(s):

A. Deisseroth ◽

J. Barker ◽

W. F. Anderson ◽

Nienhuis

Keyword(s):

Somatic Cell ◽

Chinese Hamster ◽

Globin Genes ◽

Somatic Cell Hybrids ◽

Hemoglobin Synthesis ◽

Cell Hybrids ◽

Erythroleukemia Cells ◽

Mouse Erythroleukemia

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Comparison of Expression of Human Globin Genes Transferred Into Mouse Erythroleukemia Cells and in Transgenic Mice

Blood ◽

10.1182/blood.v92.9.3416.421k16_3416_3421 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3416-3421 ◽

Cited By ~ 2

Author(s):

E. Skarpidi ◽

G. Vassilopoulos ◽

G. Stamatoyannopoulos ◽

Q. Li

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Globin Gene ◽

Mrna Levels ◽

Globin Genes ◽

Position Effects ◽

Erythroleukemia Cells ◽

Cell Clones ◽

Globin Gene Expression ◽

Mouse Erythroleukemia

To examine whether transfer of γ globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of γ globin gene promoter, Aγ gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (μLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Aγ globin gene expression among MEL cell clones carrying the μLCR(−201)Aγ and μLCR(−382)Aγ gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between theAγ mRNA levels and the copies of the transgene (r= .28, P = .18). There was significant variation in per copy Aγ globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Aγ globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study ofcis elements controlling γ globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects. © 1998 by The American Society of Hematology.

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Regulation of human embryonic globin genes zeta 2 and epsilon in stably transformed mouse erythroleukemia cells

Blood ◽

10.1182/blood.v80.7.1832.bloodjournal8071832 ◽

1992 ◽

Vol 80 (7) ◽

pp. 1832-1837 ◽

Cited By ~ 1

Author(s):

P Vyas ◽

JA Sharpe ◽

P Watt ◽

DR Higgs ◽

WG Wood

Keyword(s):

Regulatory Elements ◽

Erythroid Cell ◽

Regulatory Sequences ◽

Globin Genes ◽

Promoter Regions ◽

Regulatory Regions ◽

Chromosomal Organization ◽

Erythroleukemia Cells ◽

Cast Doubt ◽

Mouse Erythroleukemia

Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.

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Regulation of human embryonic globin genes zeta 2 and epsilon in stably transformed mouse erythroleukemia cells

Blood ◽

10.1182/blood.v80.7.1832.1832 ◽

1992 ◽

Vol 80 (7) ◽

pp. 1832-1837 ◽

Cited By ~ 10

Author(s):

P Vyas ◽

JA Sharpe ◽

P Watt ◽

DR Higgs ◽

WG Wood

Keyword(s):

Regulatory Elements ◽

Erythroid Cell ◽

Regulatory Sequences ◽

Globin Genes ◽

Promoter Regions ◽

Regulatory Regions ◽

Chromosomal Organization ◽

Erythroleukemia Cells ◽

Cast Doubt ◽

Mouse Erythroleukemia

Abstract Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.

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Comparison of Expression of Human Globin Genes Transferred Into Mouse Erythroleukemia Cells and in Transgenic Mice

Blood ◽

10.1182/blood.v92.9.3416 ◽

1998 ◽

Vol 92 (9) ◽

pp. 3416-3421 ◽

Cited By ~ 15

Author(s):

E. Skarpidi ◽

G. Vassilopoulos ◽

G. Stamatoyannopoulos ◽

Q. Li

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Globin Gene ◽

Mrna Levels ◽

Globin Genes ◽

Position Effects ◽

Erythroleukemia Cells ◽

Cell Clones ◽

Globin Gene Expression ◽

Mouse Erythroleukemia

Abstract To examine whether transfer of γ globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of γ globin gene promoter, Aγ gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (μLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Aγ globin gene expression among MEL cell clones carrying the μLCR(−201)Aγ and μLCR(−382)Aγ gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between theAγ mRNA levels and the copies of the transgene (r= .28, P = .18). There was significant variation in per copy Aγ globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Aγ globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study ofcis elements controlling γ globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects. © 1998 by The American Society of Hematology.

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Regulation of human fetal and adult globin genes in mouse erythroleukemia cells

Blood ◽

10.1182/blood.v78.5.1355.bloodjournal7851355 ◽

1991 ◽

Vol 78 (5) ◽

pp. 1355-1363 ◽

Cited By ~ 1

Author(s):

BJ Morley ◽

CA Abbott ◽

WG Wood

Keyword(s):

Globin Genes ◽

Beta Globin ◽

Chromosome 11 ◽

Globin Mrna ◽

Chromosomal Organization ◽

Gamma Globin ◽

Erythroleukemia Cells ◽

Regulatory Information ◽

Beta Gene ◽

Mouse Erythroleukemia

We have examined whether transfected mouse erythroleukaemia (MEL) cells can be used to examine differential expression of human gamma- and beta- globin genes. These cells, which express only their adult globin genes, will transcribe the human adult beta gene but not the fetal gamma genes when they are introduced on an intact human chromosome 11 by cell fusion. However, MEL cells stably transfected with the human A gamma gene attached to one of the active elements (HS2) of the beta-globin locus control region (LCR) readily produce gamma-globin mRNA in amounts equivalent to those seen with a comparable beta gene insert. When both beta and gamma genes are attached to HS2, equal amounts of beta A gamma mRNAs are produced, irrespective of the gene order. Furthermore, when HS2 is inserted into the 5′ end of a 40-kb cosmid containing the G gamma A gamma-117 delta beta genes in their normal chromosomal organization (but with the Greek HPFH -117 A gamma gene mutation), it directs expression of readily detectable amounts of G gamma A gamma and beta-globin mRNAs in MEL cells. Therefore, under these circumstances we have observed no competition between beta and gamma genes for expression in MEL cells. These findings suggest that MEL cells are capable of perpetuating regulatory information involved in developmental control when it is provided by an intact chromosome, but are incapable of reconstructing such information on transfected DNA.

Download Full-text