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. 1986 Jun;83(12):4312–4316. doi: 10.1073/pnas.83.12.4312

Regulated gene expression in transfected primary chicken erythrocytes.

J E Hesse, J M Nickol, M R Lieber, G Felsenfeld
PMCID: PMC323722  PMID: 3459175

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bruns G. A., Ingram V. M. The erythroid cells and haemoglobins of the chick embryo. Philos Trans R Soc Lond B Biol Sci. 1973 Oct 25;266(877):225–305. doi: 10.1098/rstb.1973.0050. [DOI] [PubMed] [Google Scholar]
  2. Camerini-Otero R. D., Zasloff M. A. Nucleosomal packaging of the thymidine kinase gene of herpes simplex virus transferred into mouse cells: an actively expressed single-copy gene. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5079–5083. doi: 10.1073/pnas.77.9.5079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Charnay P., Treisman R., Mellon P., Chao M., Axel R., Maniatis T. Differences in human alpha- and beta-globin gene expression in mouse erythroleukemia cells: the role of intragenic sequences. Cell. 1984 Aug;38(1):251–263. doi: 10.1016/0092-8674(84)90547-6. [DOI] [PubMed] [Google Scholar]
  4. Dolan M., Dodgson J. B., Engel J. D. Analysis of the adult chicken beta-globin gene. Nucleotide sequence of the locus, microheterogeneity at the 5'-end of beta-globin mRNA, and aberrant nuclear RNA species. J Biol Chem. 1983 Mar 25;258(6):3983–3990. [PubMed] [Google Scholar]
  5. Emerson B. M., Felsenfeld G. Specific factor conferring nuclease hypersensitivity at the 5' end of the chicken adult beta-globin gene. Proc Natl Acad Sci U S A. 1984 Jan;81(1):95–99. doi: 10.1073/pnas.81.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Emerson B. M., Lewis C. D., Felsenfeld G. Interaction of specific nuclear factors with the nuclease-hypersensitive region of the chicken adult beta-globin gene: nature of the binding domain. Cell. 1985 May;41(1):21–30. doi: 10.1016/0092-8674(85)90057-1. [DOI] [PubMed] [Google Scholar]
  7. Ginder G. D., Wood W. I., Felsenfeld G. Isolation and characterization of recombinant clones containing the chicken adult beta-globin gene. J Biol Chem. 1979 Sep 10;254(17):8099–8102. [PubMed] [Google Scholar]
  8. Gorman C. M., Merlino G. T., Willingham M. C., Pastan I., Howard B. H. The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6777–6781. doi: 10.1073/pnas.79.22.6777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jackson P. D., Felsenfeld G. A method for mapping intranuclear protein-DNA interactions and its application to a nuclease hypersensitive site. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2296–2300. doi: 10.1073/pnas.82.8.2296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laimins L. A., Tsichlis P., Khoury G. Multiple enhancer domains in the 3' terminus of the Prague strain of Rous sarcoma virus. Nucleic Acids Res. 1984 Aug 24;12(16):6427–6442. doi: 10.1093/nar/12.16.6427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lieber M. R., Steck T. L. A description of the holes in human erythrocyte membrane ghosts. J Biol Chem. 1982 Oct 10;257(19):11651–11659. [PubMed] [Google Scholar]
  13. Lopata M. A., Cleveland D. W., Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res. 1984 Jul 25;12(14):5707–5717. doi: 10.1093/nar/12.14.5707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  15. McCutchan J. H., Pagano J. S. Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J Natl Cancer Inst. 1968 Aug;41(2):351–357. [PubMed] [Google Scholar]
  16. McGhee J. D., Wood W. I., Dolan M., Engel J. D., Felsenfeld G. A 200 base pair region at the 5' end of the chicken adult beta-globin gene is accessible to nuclease digestion. Cell. 1981 Nov;27(1 Pt 2):45–55. doi: 10.1016/0092-8674(81)90359-7. [DOI] [PubMed] [Google Scholar]
  17. Stalder J., Groudine M., Dodgson J. B., Engel J. D., Weintraub H. Hb switching in chickens. Cell. 1980 Apr;19(4):973–980. doi: 10.1016/0092-8674(80)90088-4. [DOI] [PubMed] [Google Scholar]
  18. Stalder J., Larsen A., Engel J. D., Dolan M., Groudine M., Weintraub H. Tissue-specific DNA cleavages in the globin chromatin domain introduced by DNAase I. Cell. 1980 Jun;20(2):451–460. doi: 10.1016/0092-8674(80)90631-5. [DOI] [PubMed] [Google Scholar]
  19. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  20. Wright S., Rosenthal A., Flavell R., Grosveld F. DNA sequences required for regulated expression of beta-globin genes in murine erythroleukemia cells. Cell. 1984 Aug;38(1):265–273. doi: 10.1016/0092-8674(84)90548-8. [DOI] [PubMed] [Google Scholar]

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