Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells

Koller, B H; Kim, Hyungsuk; Latour, A M; Brigman, K; Boucher, Jr, R C; Smithies, O; Scambler, P; Wainwright, B

doi:10.1073/pnas.88.23.10730

Title: Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells

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Abstract

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possible passage number, influences the recovery of CFTR-targeted cells.

Authors:

Koller, B H; Kim, Hyungsuk; Latour, A M; Brigman, K; Boucher, Jr, R C; Smithies, O ^[1]; Scambler, P; Wainwright, B ^[2]

Univ. of North Carolina, Chapel Hill (United States)
St. Mary's Hospital, London (England)

Publication Date:: Sun Dec 01 00:00:00 EST 1991

OSTI Identifier:: 5701708

Resource Type:: Journal Article

Journal Name:: Proceedings of the National Academy of Sciences of the United States of America; (United States)

Additional Journal Information:: Journal Volume: 88:23; Journal ID: ISSN 0027-8424

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; EMBRYONIC CELLS; GENE RECOMBINATION; FIBROSIS; BIOLOGICAL MODELS; HEREDITARY DISEASES; ETIOLOGY; BACTERIOPHAGES; GENOME MUTATIONS; HERPES SIMPLEX; HUMAN POPULATIONS; MICE; PATIENTS; PHENYLALANINE; PHOSPHOTRANSFERASES; STEM CELLS; AMINO ACIDS; ANIMAL CELLS; ANIMALS; CARBOXYLIC ACIDS; DISEASES; ENZYMES; INFECTIOUS DISEASES; MAMMALS; MICROORGANISMS; MUTATIONS; ORGANIC ACIDS; ORGANIC COMPOUNDS; PARASITES; PATHOLOGICAL CHANGES; PHOSPHORUS-GROUP TRANSFERASES; POPULATIONS; PROTEINS; RODENTS; SKIN DISEASES; SOMATIC CELLS; TRANSFERASES; VERTEBRATES; VIRAL DISEASES; VIRUSES; 550400* - Genetics

Citation Formats


                    Koller, B H, Kim, Hyungsuk, Latour, A M, Brigman, K, Boucher, Jr, R C, Smithies, O, Scambler, P, and Wainwright, B. Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells.  United States: N. p., 1991. 
        Web.  doi:10.1073/pnas.88.23.10730.

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                    Koller, B H, Kim, Hyungsuk, Latour, A M, Brigman, K, Boucher, Jr, R C, Smithies, O, Scambler, P, & Wainwright, B. Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells.  United States.  https://doi.org/10.1073/pnas.88.23.10730

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                    Koller, B H, Kim, Hyungsuk, Latour, A M, Brigman, K, Boucher, Jr, R C, Smithies, O, Scambler, P, and Wainwright, B. 1991.  
        "Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells".  United States.  https://doi.org/10.1073/pnas.88.23.10730.

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@article{osti_5701708,

  title        = {Toward an animal model of cystic fibrosis: Targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells},

  author       = {Koller, B H and Kim, Hyungsuk and Latour, A M and Brigman, K and Boucher, Jr, R C and Smithies, O and Scambler, P and Wainwright, B},

  abstractNote = {A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence). A herpes simplex thymidine kinase gene was on each end of the construct, which was electroporated into embryonic stem (ES) cells. Colonies resistant to G418, or to G418 plus ganciclovir, were selected and screened by Southern blotting or by PCR amplification. Five pools of G418-resistant cells gave PCR products diagnostic of targeting. Four independent clones of ES cells with a disrupted CFTR gene have been isolated from these pools. The frequency of targeting was 1/2500 G418-resistant colonies. This low frequency is not the consequence of marginal expression of the Neo genes in the targeted cells. The CFTR targeting events were clustered among our experiments in a manner suggesting that some unidentified factor(s), possible passage number, influences the recovery of CFTR-targeted cells.},

  doi          = {10.1073/pnas.88.23.10730},

  url          = {https://www.osti.gov/biblio/5701708},
  journal      = {Proceedings of the National Academy of Sciences of the United States of America; (United States)},

  issn         = {0027-8424},

  number       = ,

  volume       = 88:23,

  place        = {United States},

  year         = {Sun Dec 01 00:00:00 EST 1991},

  month        = {Sun Dec 01 00:00:00 EST 1991}

}

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