Insertion of DNA sequences into the human chromosomal β-globin locus by homologous recombination

  title={Insertion of DNA sequences into the human chromosomal $\beta$-globin locus by homologous recombination},
  author={Oliver Smithies and Ronald G. Gregg and Sallie S. Boggs and Michał Koralewski and Raju Kucherlapati},
A ‘rescuable’ plasmid containing globin gene sequences allowing recombination with homologous chromosomal sequences has enabled us to produce, score and clone mammalian cells with the plasmid integrated into the human β-globin locus. The planned modification was achieved in about one per thousand transformed cells whether or not the target gene was expressed. 
Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene
Injection of homologous DNA sequences into nuclei of cultured mammalian cells induces mutations in the cognate chromosomal gene, which suggests that this method may prove useful for introducing mutations into specific mammalian genes.
Accurate modification of a chromosomal plasmid by homologous recombination in human cells.
Analyses of the functional neo genes in various independent cell lines establish that simple crossovers generated the wild-type neo genes.
Intrachromosomal Recombination in Mammalian Cells
The technologies of gene transfer and recombinant DNA can be exploited to construct artificial gene duplications of a selectable gene in cultured mammalian cells to systematically study the process of homologous recombination as it occurs between repeated chromosomal sequences in cultured mammals cells.
Analysis of mutations introduced into the chromosomal immunoglobulin μ gene
Analysis of the DNA in independent noncytolytic transformants indicates that in one case the μ gene has the structure expected for correct homologous recombination, and unexpectedly, the remaining transformants, bear chromosomal μ gene deletions.
Improving gene replacement by intracellular formation of linear homologous DNA
This work has developed a system for the intracellular release of linear fragments from circular plasmids, which is more efficient than transfection of linear DNA and adaptable to viral vectors.
Homologous recombination in a mammalian plasmid
Sequencing after rescue of the plasmid inEscherichia coli showed that most of the recombinants were from accurate homologous recombination, and possible mechanisms that explain these features are discussed.
Chromosome accommodation to integration of foreign DNA
The discovery of covalent linkage between chromosomal DNA and DNA introduced by DNA-mediated transfer led to new approaches for gene mapping and studies of gene regulation in mammalian cells.
Gene targeting with retroviral vectors: recombination by gene conversion into regions of nonhomology
It is demonstrated that retroviral vectors can recombine with homologous chromosomal sequences in rodent and human cells.
Using Homologous Recombination to Manipulate the Genome of Human Somatic Cells
  • M. Porteus
  • Biology
    Biotechnology & genetic engineering reviews
  • 2007
Abbreviations: rAAV, recombinant adeno-associated virus; DNA, deoxyribonucleic acid; DSBs, doublestrand breaks; SDSA, synthesis dependent strand annealing; ES, embryonic stem; ESC, embryonic stem


Purification of genomic sequences from bacteriophage libraries by recombination and selection in vivo.
  • B. Seed
  • Biology
    Nucleic acids research
  • 1983
Cloned genes have been purified from recombinant DNA bacteriophage libraries by a method exploiting homologous reciprocal recombination in vivo. In this method 'probe' sequences are inserted in a
Escherichia coli extract-catalyzed recombination in switch regions of mouse immunoglobulin genes.
We have shown that Escherichia coli extracts catalyze recombination between mouse immunoglobulin mu and alpha genes inserted separately in lambda phage vectors carrying different genetic markers.
Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA.
We have used the eukaryotic-prokaryotic shuttle vector pSV2Neo to demonstrate that cultured mammalian somatic cells have the enzymatic machinery to mediate homologous recombination and that the
Yeast transformation: a model system for the study of recombination.
Consideration of models for plasmid integration and gene conversion suggests that RAD52 may be involved in the DNA repair synthesis required for these processes and implications for the isolation of integrative transformants, fine-structure mapping, and the cloning of mutations are discussed.
Transformation of yeast.
This work has used recently developed hybridization and restriction endonuclease mapping techniques to demonstrate directly the presence of the transforming DNA in the yeast genome and also to determine the arrangement of the sequences that were introduced.
Recombination in mouse L cells between DNA introduced into cells and homologous chromosomal sequences.
It is shown that DNA added to mouse L cells by the calcium phosphate method can be inserted into the genome of those cells by homologous recombination, and that relatively little illegitimate insertion of introduced tk DNA into cellular DNA was detected in those cells that were transformed to Tk+ by homologies recombination.
A transmissible retrovirus expressing human hypoxanthine phosphoribosyltransferase (HPRT): gene transfer into cells obtained from humans deficient in HPRT.
A cDNA corresponding to the human gene for hypoxanthine phosphoribosyltransferase has been ligated into murine retroviral vectors such that it is under the transcriptional control of viral long terminal repeats.
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These studies demonstrate that regulatory sequences closely linked to the β-globin gene are sufficient to specify a correct pattern of tissue-specific expression in a developing mouse, when the gene is integrated at a subset of foreign chromosomal positions.
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The most plausible explanation for transformation with the cloned class I genes of the major histocompatibility complex of the mouse express transplantation antigens with serological determinants of the donor haplotype involves homologous recombination between host chromosomal and donor class I sequences.