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.

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.

Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination

Recombination between chromosomal and extrachromosomal DNA sequences was analyzed by investigation of the recombinational rescue of a 1,018-base-pair (bp) segment of the T-antigen gene of simian

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.

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.

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.

Specific expression of a foreign β-globin gene in erythroid cells of transgenic mice

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.

Genetic transformation of mouse embryos by microinjection of purified DNA.

The results demonstrate that genes can be introduced into the mouse genome by direct insertion into the nuclei of early embryos by microinjected into pronuclei of fertilized mouse oocytes.

In vitro packaging of lambda and cosmid DNA.

  • B. Hohn
  • Biology
    Methods in enzymology
  • 1979

Transcripts of human HLA gene fragments lacking the 5'-terminal region in transfected mouse cells.

Clones of mouse L cells transfected with a human HLA-B7 gene fragment lacking the 5' segment of exon 2 and all upstream sequences express Hla-specific transcripts of various lengths indicate that the level of these transcripts is increased in cells treated with interferon.