Author
Michele P. Calos
Other affiliations: Howard Hughes Medical Institute, Harvard University, University of Geneva
Bio: Michele P. Calos is an academic researcher from Stanford University. The author has contributed to research in topics: Integrase & Plasmid. The author has an hindex of 50, co-authored 148 publications receiving 11099 citations. Previous affiliations of Michele P. Calos include Howard Hughes Medical Institute & Harvard University.
Topics: Integrase, Plasmid, Recombinase, Transgene, Gene
Papers published on a yearly basis
Papers
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TL;DR: The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea and provide support for the mutational theory of cancer.
Abstract: We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.
1,137 citations
TL;DR: The phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring.
Abstract: The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.
1,073 citations
TL;DR: Using lacl-Z fusion strains of Escherichia coli, systems that detect deletions of varying lengths are devised and possible mechanisms of deletion formation are discussed and its relationship to the excision of transposable elements is considered.
619 citations
TL;DR: It is demonstrated that in the presence of the phiC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells, forming the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.
Abstract: The integrase from the Streptomyces phage φC31 carries out efficient recombination between the attP site in the phage genome and the attB site in the host bacterial chromosome. In this paper, we show that the enzyme also functions in human cells. A plasmid assay system was constructed that measured intramolecular integration of attP into attB. This assay was used to demonstrate that in the presence of the φC31 integrase, precise unidirectional integration occurs with an efficiency of 100% in Escherichia coli and >50% in human cells. This assay system was also used to define the minimal sizes of attB and attP at 34 bp and 39 bp, respectively. Furthermore, precise and efficient intermolecular integration of an incoming plasmid bearing attP into an established Epstein–Barr virus plasmid bearing attB was documented in human cells. This work is a demonstration of efficient, site-specific, unidirectional integration in mammalian cells. These observations form the basis for site-specific integration strategies potentially useful in a broad range of genetic engineering applications.
558 citations
TL;DR: Integrases of the serine family have been shown to work efficiently in mammalian cells, mediating efficient integration at introduced att sites or native sequences that have partial identity to att sites, which has applications in areas such as gene therapy, construction of transgenic organisms, and manipulation of cell lines.
514 citations
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7,047 citations
TL;DR: Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum.
6,003 citations
TL;DR: Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells and revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide with a single transmembrane domain.
2,918 citations
TL;DR: In this article, a method for producing high-titer, helper-free infectious retroviruses is disclosed which employs a novel strategy that uses transient transfection of new retroviral producer cell lines, ecotropic line BOSC 23 and amphotropic line CAK 8.
Abstract: A method for producing high-titer, helper-free infectious retroviruses is disclosed which employs a novel strategy that uses transient transfection of new retroviral producer cell lines, ecotropic line BOSC 23 and amphotropic line CAK 8, both of which cell lines and their precursor cell lines are disclosed. Because of the advantages over stable packaging cell lines, the BOSC 23 and CAK 8 transient transfection systems greatly facilitate and extend the use of helper-free retroviral vectors. The cell lines and corresponding methods possess wide application in both the medical and biotechnical fields, including gene therapy. These potential applications are disclosed and illustrated.
2,587 citations
TL;DR: With the development of a leukaemia-like syndrome in two patients cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.
Abstract: Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.
2,451 citations