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PMID: 2349238 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 87 ·No. 11 ·1990-06-00 ·Pages 4299-303

Reid LH, Gregg RG, Smithies O, Koller BH

Abstract

We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene.

MeSH Terms
Animals Cell Line Cloning, Molecular DNA/genetics DNA Mutational Analysis Embryo, Mammalian/physiology Enhancer Elements, Genetic Gene Expression Regulation Hypoxanthine Phosphoribosyltransferase/genetics Introns Mice Regulatory Sequences, Nucleic Acid Restriction Mapping Stem Cells/physiology
Chemicals
DNA Hypoxanthine Phosphoribosyltransferase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Reid L H
Department of Pathology, University of North Carolina, Chapel Hill 27599.
Gregg R G
Smithies O
Koller B H
References (30)
30 references, click to expand
  1. The organization of the human HPRT gene.
    Nucleic Acids Res. 1986 Apr 11;14(7):3103-18 PMID: 3008106
  2. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium.
    J Embryol Exp Morphol. 1985 Jun;87:27-45 PMID: 3897439
  3. Fine structure of the human hypoxanthine phosphoribosyltransferase gene.
    Mol Cell Biol. 1986 Feb;6(2):393-403 PMID: 3023844
  4. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells.
    Nature. 1987 Mar 19-25;326(6110):292-5 PMID: 3821905
  5. Rat prostatic binding protein: the complete sequence of the C2 gene and its flanking regions.
    Nucleic Acids Res. 1987 Feb 25;15(4):1627-41 PMID: 2881277
  6. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.
    Cell. 1987 Nov 6;51(3):503-12 PMID: 2822260
  7. Rapid and efficient site-specific mutagenesis without phenotypic selection.
    Methods Enzymol. 1987;154:367-82 PMID: 3323813
  8. Introns increase transcriptional efficiency in transgenic mice.
    Proc Natl Acad Sci U S A. 1988 Feb;85(3):836-40 PMID: 3422466
  9. Recombinant fragment assay for gene targetting based on the polymerase chain reaction.
    Nucleic Acids Res. 1988 Sep 26;16(18):8887-903 PMID: 3174435
  10. Comparison of intron-dependent and intron-independent gene expression.
    Mol Cell Biol. 1988 Oct;8(10):4395-405 PMID: 3185553
  11. Targeted mutation of the Hprt gene in mouse embryonic stem cells.
    Proc Natl Acad Sci U S A. 1988 Nov;85(22):8583-7 PMID: 3186749
  12. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes.
    Nature. 1988 Nov 24;336(6197):348-52 PMID: 3194019
  13. 5' flanking and first intron sequences of the human beta-actin gene required for efficient promoter activity.
    Nucleic Acids Res. 1989 Jan 11;17(1):253-70 PMID: 2911466
  14. Importance of introns for expression of mouse ribosomal protein gene rpL32.
    Mol Cell Biol. 1989 May;9(5):2075-82 PMID: 2747643
  15. Regulatory elements in the 5'-flanking region and the first intron contribute to transcriptional control of the mouse alpha 1 type I collagen gene.
    Mol Cell Biol. 1989 May;9(5):2224-7 PMID: 2747648
  16. The spatial and temporal expression pattern of sevenless is exclusively controlled by gene-internal elements.
    EMBO J. 1989 Aug;8(8):2381-6 PMID: 2792089
  17. Differential binding of nuclear factors to the intron 1 sequences containing the transcriptional pause site correlates with c-myb expression.
    Proc Natl Acad Sci U S A. 1989 Oct;86(19):7326-30 PMID: 2678098
  18. Germ-line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells.
    Proc Natl Acad Sci U S A. 1989 Nov;86(22):8927-31 PMID: 2573070
  19. Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells.
    Science. 1989 Nov 10;246(4931):799-803 PMID: 2554496
  20. Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells.
    Nature. 1989 Nov 23;342(6248):435-8 PMID: 2685607
  21. Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene.
    Genes Dev. 1989 Sep;3(9):1384-400 PMID: 2606352
  22. Automated DNA sequencing of the human HPRT locus.
    Genomics. 1990 Apr;6(4):593-608 PMID: 2341149
  23. Replacement of chromosome segments with altered DNA sequences constructed in vitro.
    Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951-5 PMID: 388424
  24. Multiple point mutations affecting the simian virus 40 enhancer.
    Science. 1983 Feb 11;219(4585):626-31 PMID: 6297005
  25. Isolation and characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyl transferase.
    Proc Natl Acad Sci U S A. 1983 Jan;80(2):477-81 PMID: 6300847
  26. The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element.
    Cell. 1983 Jul;33(3):695-703 PMID: 6871991
  27. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes.
    Cell. 1983 Jul;33(3):729-40 PMID: 6409418
  28. DNA fragments from F9 PyEC mutants increase expression of heterologous genes in transfected F9 cells.
    Cell. 1983 Dec;35(3 Pt 2):693-9 PMID: 6317199
  29. Structure, expression, and mutation of the hypoxanthine phosphoribosyltransferase gene.
    Proc Natl Acad Sci U S A. 1984 Apr;81(7):2147-51 PMID: 6326107
  30. Efficient transformation and frequent single-site, single-copy insertion of DNA can be obtained in mouse erythroleukemia cells transformed by electroporation.
    Exp Hematol. 1986 Nov;14(10):988-94 PMID: 3464450
Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.87.11.4299
Published
1990-06-00
Pages
4299-303
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC54096
Subset
IM
Grants
NIGMS NIH HHS · GM20069 · United States
NHLBI NIH HHS · HL37001 · United States
External Links
PubMed source DOI: 10.1073/pnas.87.11.4299 PMC Full Text View journal details
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