• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Jun 1990; 87(11): 4299–4303.
PMCID: PMC54096

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

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.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Koller BH, Hagemann LJ, Doetschman T, Hagaman JR, Huang S, Williams PJ, First NL, Maeda N, Smithies O. 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):8927–8931. [PMC free article] [PubMed]
  • Zijlstra M, Li E, Sajjadi F, Subramani S, Jaenisch R. Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells. Nature. 1989 Nov 23;342(6248):435–438. [PubMed]
  • Schwartzberg PL, Goff SP, Robertson EJ. Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science. 1989 Nov 10;246(4931):799–803. [PubMed]
  • Mansour SL, Thomas KR, Capecchi MR. 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):348–352. [PubMed]
  • Doetschman T, Maeda N, Smithies O. Targeted mutation of the Hprt gene in mouse embryonic stem cells. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8583–8587. [PMC free article] [PubMed]
  • Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987 Nov 6;51(3):503–512. [PubMed]
  • Melton DW, Konecki DS, Brennand J, Caskey CT. Structure, expression, and mutation of the hypoxanthine phosphoribosyltransferase gene. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2147–2151. [PMC free article] [PubMed]
  • Patel PI, Framson PE, Caskey CT, Chinault AC. Fine structure of the human hypoxanthine phosphoribosyltransferase gene. Mol Cell Biol. 1986 Feb;6(2):393–403. [PMC free article] [PubMed]
  • Kim SH, Moores JC, David D, Respess JG, Jolly DJ, Friedmann T. The organization of the human HPRT gene. Nucleic Acids Res. 1986 Apr 11;14(7):3103–3118. [PMC free article] [PubMed]
  • Frederickson RM, Micheau MR, Iwamoto A, Miyamoto NG. 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):253–270. [PMC free article] [PubMed]
  • Banerji J, Olson L, Schaffner W. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell. 1983 Jul;33(3):729–740. [PubMed]
  • Aronow B, Lattier D, Silbiger R, Dusing M, Hutton J, Jones G, Stock J, McNeish J, Potter S, Witte D, et al. Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene. Genes Dev. 1989 Sep;3(9):1384–1400. [PubMed]
  • Rippe RA, Lorenzen SI, Brenner DA, Breindl M. 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):2224–2227. [PMC free article] [PubMed]
  • Basler K, Siegrist P, Hafen E. The spatial and temporal expression pattern of sevenless is exclusively controlled by gene-internal elements. EMBO J. 1989 Aug;8(8):2381–2386. [PMC free article] [PubMed]
  • Reddy CD, Reddy EP. 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):7326–7330. [PMC free article] [PubMed]
  • Chung S, Perry RP. Importance of introns for expression of mouse ribosomal protein gene rpL32. Mol Cell Biol. 1989 May;9(5):2075–2082. [PMC free article] [PubMed]
  • Buchman AR, Berg P. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol. 1988 Oct;8(10):4395–4405. [PMC free article] [PubMed]
  • Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD. Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci U S A. 1988 Feb;85(3):836–840. [PMC free article] [PubMed]
  • Hooper M, Hardy K, Handyside A, Hunter S, Monk M. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature. 1987 Mar 19;326(6110):292–295. [PubMed]
  • Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. 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. [PubMed]
  • Boggs SS, Gregg RG, Borenstein N, Smithies O. 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):988–994. [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Linney E, Donerly S. DNA fragments from F9 PyEC mutants increase expression of heterologous genes in transfected F9 cells. Cell. 1983 Dec;35(3 Pt 2):693–699. [PubMed]
  • Jolly DJ, Okayama H, Berg P, Esty AC, Filpula D, Bohlen P, Johnson GG, Shively JE, Hunkapillar T, Friedmann T. 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):477–481. [PMC free article] [PubMed]
  • Edwards A, Voss H, Rice P, Civitello A, Stegemann J, Schwager C, Zimmermann J, Erfle H, Caskey CT, Ansorge W. Automated DNA sequencing of the human HPRT locus. Genomics. 1990 Apr;6(4):593–608. [PubMed]
  • Hearing P, Shenk T. The adenovirus type 5 E1A transcriptional control region contains a duplicated enhancer element. Cell. 1983 Jul;33(3):695–703. [PubMed]
  • Weiher H, König M, Gruss P. Multiple point mutations affecting the simian virus 40 enhancer. Science. 1983 Feb 11;219(4585):626–631. [PubMed]
  • Delaey B, Dirckx L, Decourt JL, Claessens F, Peeters B, Rombauts W. Rat prostatic binding protein: the complete sequence of the C2 gene and its flanking regions. Nucleic Acids Res. 1987 Feb 25;15(4):1627–1641. [PMC free article] [PubMed]
  • Kim HS, Smithies O. Recombinant fragment assay for gene targetting based on the polymerase chain reaction. Nucleic Acids Res. 1988 Sep 26;16(18):8887–8903. [PMC free article] [PubMed]
  • Scherer S, Davis RW. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...