Targeted mutation of the Hprt gene in mouse embryonic stem cells

Thomas C Doetschman, N. Maeda, O. Smithies

Research output: Contribution to journalArticle

172 Citations (Scopus)

Abstract

The hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene has been mutated in mouse blastocyst-derived embryonic stem cells by site-directed homologous recombination. Embryonic stem cells were electroporated in the presence of a targeting DNA fragment containing two specific features: (i) The targeting DNA contained a promoterless neomycin phosphotransferase (neo) gene that, when located within the endogenous Hprt locus, could be transcribed from the promoter of the target locus. (ii) The targeting fragment had two short regions of homology with the endogenous Hprt gene: one, 132 base pairs long and the other, 1.2 kilobase pairs long. Targeted cells in which the designed homologous recombination event occurred were isolated either by selection with G418 followed by 6-thioguanine or by selection with 6-thioguanine alone. Even though <2 kilobases of homology existed between the exogenous and target DNAs, an average of 2.6 embryonic stem cells were successfully targeted for every 105 colonies surviving electroporation. Six of the Hprt- cell lines showed homologous recombination. These six lines were further analyzed by nucleotide sequencing a fragment that spans one crossover point after amplification by the polymerase chain reaction. Four lines had the expected sequence, whereas two lines had small deletions abutting the 132-base-pair region of homology.

Original languageEnglish (US)
Pages (from-to)8583-8587
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number22
StatePublished - 1988
Externally publishedYes

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Hypoxanthine Phosphoribosyltransferase
Homologous Recombination
Embryonic Stem Cells
Thioguanine
Mutation
Base Pairing
Genes
DNA
Kanamycin Kinase
Electroporation
Blastocyst
Nucleotides
Cell Line
Polymerase Chain Reaction
Mouse Embryonic Stem Cells

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

Targeted mutation of the Hprt gene in mouse embryonic stem cells. / Doetschman, Thomas C; Maeda, N.; Smithies, O.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, No. 22, 1988, p. 8583-8587.

Research output: Contribution to journalArticle

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AB - The hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene has been mutated in mouse blastocyst-derived embryonic stem cells by site-directed homologous recombination. Embryonic stem cells were electroporated in the presence of a targeting DNA fragment containing two specific features: (i) The targeting DNA contained a promoterless neomycin phosphotransferase (neo) gene that, when located within the endogenous Hprt locus, could be transcribed from the promoter of the target locus. (ii) The targeting fragment had two short regions of homology with the endogenous Hprt gene: one, 132 base pairs long and the other, 1.2 kilobase pairs long. Targeted cells in which the designed homologous recombination event occurred were isolated either by selection with G418 followed by 6-thioguanine or by selection with 6-thioguanine alone. Even though <2 kilobases of homology existed between the exogenous and target DNAs, an average of 2.6 embryonic stem cells were successfully targeted for every 105 colonies surviving electroporation. Six of the Hprt- cell lines showed homologous recombination. These six lines were further analyzed by nucleotide sequencing a fragment that spans one crossover point after amplification by the polymerase chain reaction. Four lines had the expected sequence, whereas two lines had small deletions abutting the 132-base-pair region of homology.

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