Correction of human. beta. sup S -globin gene by gene targeting

Journal Article · Wed May 15 00:00:00 EDT 1991 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.88.10.4294· OSTI ID:5934060

Shesely, E G; Kim, Hyungsuk; Shehee, W R; Smithies, O ^[1]; Papayannopoulou, T ^[2]; Popovich, B W ^[3]

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1. Univ. of North Carolina, Chapel Hill (United States)
2. Univ. of Washington, Seattle (United States)
3. Children's Hospital, San Diego, CA (United States)

As a step toward using gene targeting for gene therapy, the authors have corrected a human {beta}{sup S}-globin gene to the normal {beta}{sup A} allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the {beta}{sup S}-globin allele. A {beta}{sup A}-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total {approx}29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the {beta}{sup S} allele to {beta}{sup A} was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5{prime} to the human {beta}{sup A}-globin gene. Thus gene targeting can correct a {beta}{sup S} allele to {beta}{sup A}, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

• OSTI ID: 5934060
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 88:10; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
GLOBINS
GENE REGULATION
SICKLE CELL ANEMIA
THERAPY
GENE RECOMBINATION
GENETIC ENGINEERING
HETEROCHROMOSOMES
HYBRIDIZATION
LEUKEMIA
MICE
OLIGONUCLEOTIDES
TUMOR CELLS
ANEMIAS
ANIMAL CELLS
ANIMALS
BIOTECHNOLOGY
CHROMOSOMES
DISEASES
HEMIC DISEASES
IMMUNE SYSTEM DISEASES
MAMMALS
NEOPLASMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PROTEINS
RODENTS
SYMPTOMS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques