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Results for “targeted integration”

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At least 55 records · Page 3Linked to original sources
PubMed1992

Targeted integration of neomycin into yeast artificial chromosomes (YACs) for transfection into mammalian cells.

Vectors have been constructed for the introduction of the neomycin resistance gene (neo) into the left arm, right arm or human insert DNA of yeast artificial chromosomes (YACs) by homologous recombination. These vectors contain a yeast selectable marker Lys-2, i.e. the alpha-aminoadipidate reductase gene, and a mammalian selection marker, neo, which confers G418 resistance. The vectors can be used to modify YACs in the most commonly used yeast strain for YAC library construction, AB1380. Specific targeting can be carried out by transfection of restriction endonuclease treated linear plasmids, with highly specific recombinogenic ends, into the YAC containing yeast cells. Analysis of targeted YACs confirmed that all three vectors can target correctly in yeast. Introduction of one of the targeted YACs into V79 (Chinese hamster fibroblast) cells showed complete and intact transfer of the YAC.

Animals↗
PubMed1995

A disruption-replacement approach for the targeted integration of foreign genes in Hansenula polymorpha.

A system has been developed which allows the selection of integrative transformants with replacement of the Hansenula polymorpha methanol oxidase gene (MOX) with expression cassettes carrying heterologous gene under the control of the MOX promoter. The system is convenient for comparison of the expression levels of different constructs integrated into the same locus of the H. polymorpha genome. This system was used to compare the secretion levels of human urinary plasminogen activator, the secretion of which was directed by different signal sequences.

Alcohol Oxidoreductases↗
PubMed1991

Targeted integration of adeno-associated virus (AAV) into human chromosome 19.

A key feature in adeno-associated virus (AAV) replication is efficient integration of the viral genome into host cell DNA to establish latency when helper virus is absent. The steps involved in this process remain largely uncharacterized, even though AAV integration was first documented 20 years ago. Using a protein--DNA binding method we isolated AAV--cellular junction DNA sequences. The cellular component hybridized to a single restriction fragment in the virus-free parental cell line, and also co-migrated with AAV-specific sequences in numerous latently infected cell lines. Analysis of somatic cell hybrids indicated that this cellular sequence maps to the distal portion of the q arm of human chromosome 19. In situ hybridization of AAV DNA to chromosomes from latently infected cells confirms the physical location of AAV integrations to be q13.4-ter of chromosome 19. Sequence analysis of several independent integration sites shows breakpoints occurring within a 100 bp cellular region. This non-pathogenic parvovirus thus appears to establish viral latency by integrating its DNA specifically into one chromosomal region. Such specific integration is so far unique among the eukaryotic DNA viruses. The incorporation of site-specific integration into AAV vector schemes should make this vector system attractive for human gene therapy approaches.

Base Sequence↗
PubMed1995

Targeted integration of human herpesvirus 6 in the p arm of chromosome 17 of human peripheral blood mononuclear cells in vivo.

Out of 64 cases of non-Hodgkin's lymphomas (NHL), 55 cases of Hodgkin's disease (HD) and 31 cases of multiple sclerosis (MS), 2 NHL, 7 HD and 1 MS cases were found positive by polymerase chain reaction (PCR) for the presence of HHV-6 sequences in pathologic lymph nodes of the lymphomas and in peripheral blood mononuclear cells (PBMCs) of MS. A further analysis of the PBMCs of the PCR positive cases by standard Southern blot technique revealed only 2 NHL, 3 HD and 1 MS cases as positive, indicating that these six patients have an unusually high viral copy number in the PBMCs. Restriction analysis, carried out using probes representative of different regions of the virus, showed that three cases retain only a deleted portion of the viral genome. In the remaining three cases a complete viral genome was present, containing the right end sequences in which the rep-like gene, possibly crucial to the viral and cellular life cycle, is located. The analysis by pulsed field gel electrophoresis (PFGE) of the total DNA of the PBMCs obtained directly, without culture from PBMCs of these last three cases (1 NHL, 1 HD, and 1 MS), using the same probes, showed the absence of free viral molecules and the association of viral sequences with high molecular weight DNA. These results are consistent with in vivo integration of the entire virus in the cellular genome. A further study of the same patients with chromosome fluorescence in situ hybridization (FISH) showed in all the three cases the presence of a specific hybridization site, located at the telomeric extremity of the short arm of chromosome 17 (17p13), suggesting that this location is at least a preferred site of an infrequent, but possibly biologically important, integration phenomenon.

Adult↗
PubMed1993

Targeted integration into the Acremonium chrysogenum genome: disruption of the pcbC gene.

The cephalosporin C-producing fungus Acremonium chrysogenum was transformed to hygromycin B resistance using different vector constructs. These constructs contain sequences of the pcbC gene from A. chrysogenum, encoding isopenicillin N synthetase. Detailed analysis of transformants, including pulsed-field gel electrophoresis (PFGE), suggests that integration of multiple vector copies takes place predominantly via non-homologous integration. By increasing the length of vector-DNA homologous to genomic DNA, integration occurs more frequently into chromosome VI, carrying the endogenous pcbC gene copy. In gene disruption experiments, the length of vector homology required to obtain cephalosporin C-minus transformants was investigated. Inactivation of the pcbC gene was observed only when homologous fragments of more than 3.0 kb were used on both sites of the resistance cassette. Southern analysis indicated homologous, as well as heterologous, integration of recombinant DNA. The integration of multiple vector copies leads to the appearance of truncated pcbC transcripts.

Acremonium↗
PubMed2000

Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes.

This report describes the construction of a DNA cassette for integration into a genomic small sub-unit rRNA locus of Leishmania mexicana by homologous recombination. Reporter genes encoding beta-galactosidase or green fluorescent protein and the gene conferring hygromycin resistance were integrated downstream of a RNA polymerase I-driven rRNA promoter. To ensure high expression of the marker proteins in the intracellular, amastigote stage, transgene coding sequences were followed by the intergenic region of the L. mexicana cysteine proteinase B 2.8 gene which provides processing signals required for high level expression in this life-cycle stage. Integration of the DNA cassette was also efficiently obtained in L. major. We show that either beta-galactosidase or the green fluorescent protein were abundantly, stably and uniformly expressed in promastigotes and amastigotes of both Leishmania sp. The transgenic lines allow parasite detection at high sensitivity in the tissues of infected mice and will be useful to follow infections in macrophages in culture and in animal hosts.

Animals↗
PubMed2002

A PCR-based strategy to generate integrative targeting alleles with large regions of homology.

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

Alleles↗
PubMed1997

Denaturation of circular or linear DNA facilitates targeted integrative transformation of Streptomyces coelicolor A3(2): possible relevance to other organisms.

Using Streptomyces coelicolor A3(2) protoplasts, the number of transformants obtained by homologous recombination of incoming double-stranded circular DNA with the recipient chromosome was greatly stimulated by simple denaturation of the donor DNA. This procedure was very effective with inserts over a ca. 100-fold size range, the largest tested being ca. 40-kb inserts in cosmids. These observations led to transformation experiments with linearized cloned DNA and randomly sheared genomic DNA. In both cases, DNA denaturation led to significant levels of transformation. Most of the transformants had resulted from the predicted homologous recombination events. A number of genetic manipulations will be made easier or possible by these procedures.

Bacterial Proteins↗
PubMed1994

An integrated target sequence and signal amplification assay, reverse transcriptase-PCR-enzyme-linked immunosorbent assay, to detect and characterize flaviviruses.

We previously described a reverse transcriptase-PCR using flavivirus genus-conserved and virus species-specific amplimers (D. W. Trent and G. J. Chang, p. 355-371, in Y. Becker and C. Darai; ed., Frontiers of Virology, vol. 1, 1992). Target amplification was improved by redesigning the amplimers, and a sensitive enzyme-linked immunosorbent assay (ELISA) technique has been developed to detect amplified digoxigenin (DIG)-modified DNA. A single biotin motif and multiple DIG motifs were incorporated into each amplicon, which permitted amplicon capture by a biotin-streptavidin interaction and detection with DIG-specific antiserum in a colorimetric ELISA. We evaluated the utility of this assay for detecting St. Louis encephalitis (SLE) viral RNA in infected mosquitoes and dengue viral RNA in human serum specimens. The reverse transcriptase-PCR-ELISA was as sensitive as isolation of SLE virus by cell culture in detecting SLE viral RNA in infected mosquitoes. The test was 89% specific and 95 to 100% sensitive for identification of dengue viral RNA in serum specimens compared with isolation of virus by Aedes albopictus C6/36 cell culture and identification by the indirect immunofluorescence assay.

Aedes↗
PubMed2026

FLInt in C. briggsae and C. tropicalis : Strains and resources for fast and locus-targeted integration of multi-copy transgenes.

C. briggsae and C. tropicalis are satellite model systems for molecular mechanistic investigations and for comparative studies across Caenorhabditis species. FLInt ( F luorescent L andmark Int erference) was developed in C. elegans for rapid generation of transgenic strains through integration of multi-copy DNA arrays into predetermined genomic loci, without requiring a random mutagenesis step. Here, we present resources and newly generated strains that enable FLInt in C. briggsae and C. tropicalis , yielding integrated transgenic lines within ~10 days of microinjection.

Journal Article↗
PubMed1993

Long cellular repeats flanking a defective HTLV-I provirus: implication for site-targeted integration.

Retroviruses generally integrate as proviruses which are flanked by long-terminal repeats (LTRs) on both 5' and 3' ends. Since these LTRs are required for the efficient integration mediated by the viral integrase, it is believed that defective proviruses with a single LTR are normally formed by deletion after integration. However, we found no deletion of cellular sequences around the integration site of such a defective HTLV-1. Rather, we identified 99 bp-long direct repeats adjacent to both ends of the defective provirus. The repeated cellular sequences contained a potential poly(A) signal followed by a retroviral primer-binding-site-like sequence. The presence of the direct repeats of cellular sequences can be explained by the integration of the defective virus through homologous recombination between cellular and viral read-through sequences.

Base Sequence↗
PubMed1997

Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites.

Integration of retroviral cDNA in vivo is normally not sequence specific with respect to the integration target DNA. We have been investigating methods for directing the integration of retroviral DNA to predetermined sites, with the dual goal of understanding potential mechanisms governing normal site selection and developing possible methods for gene therapy. To this end, we have fused retroviral integrase enzymes to sequence-specific DNA-binding domains and investigated target site selection by the resulting proteins. In a previous study, we purified and analyzed a fusion protein composed of human immunodeficiency virus integrase linked to the DNA-binding domain of lambda repressor. This fusion could direct selective integration in vitro into target DNA containing lambda repressor binding sites. Here we investigate the properties of a fusion integrase in the context of a human immunodeficiency virus provirus. We used a fusion of integrase to the DNA binding domain of the zinc finger protein zif268 (IN-zif). Initially we found that the fusion was highly detrimental to replication as measured by the multinuclear activation of a galactosidase indicator (MAGI) assay for infected centers. However, we found that viruses containing mixtures of wild-type integrase and IN-zif were infectious. We prepared preintegration complexes from cells infected with these viruses and found that such complexes directed increased integration near zif268 recognition sites.

Cell Line, Transformed↗
PubMed1998

Chromosome structure and human immunodeficiency virus type 1 cDNA integration: centromeric alphoid repeats are a disfavored target.

Integration of retroviral cDNA into host chromosomal DNA is an essential and distinctive step in viral replication. Despite considerable study, the host determinants of sites for integration have not been fully clarified. To investigate integration site selection in vivo, we used two approaches. (i) We have analyzed the host sequences flanking 61 human immunodeficiency virus type 1 (HIV-1) integration sites made by experimental infection and compared them to a library of 104 control sequences. (ii) We have also analyzed HIV-1 integration frequencies near several human repeated-sequence DNA families, using a repeat-specific PCR-based assay. At odds with previous reports from smaller-scale studies, we found no strong biases either for or against integration near repetitive sequences such as Alu or LINE-1 elements. We also did not find a clear bias for integration in transcription units as proposed previously, although transcription units were found somewhat more frequently near integration sites than near controls. However, we did find that centromeric alphoid repeats were selectively absent at integration sites. The repeat-specific PCR-based assay also indicated that alphoid repeats were disfavored for integration in vivo but not as naked DNA in vitro. Evidently the distinctive DNA organization at centromeres disfavors cDNA integration. We also found a weak consensus sequence for host DNA at integration sites, and assays of integration in vitro indicated that this sequence is favored as naked DNA, revealing in addition an influence of target primary sequence.

Binding Sites↗
PubMed2006

Selection of target sites for mobile DNA integration in the human genome.

DNA sequences from retroviruses, retrotransposons, DNA transposons, and parvoviruses can all become integrated into the human genome. Accumulation of such sequences accounts for at least 40% of our genome today. These integrating elements are also of interest as gene-delivery vectors for human gene therapy. Here we present a comprehensive bioinformatic analysis of integration targeting by HIV, MLV, ASLV, SFV, L1, SB, and AAV. We used a mathematical method which allowed annotation of each base pair in the human genome for its likelihood of hosting an integration event by each type of element, taking advantage of more than 200 types of genomic annotation. This bioinformatic resource documents a wealth of new associations between genomic features and integration targeting. The study also revealed that the length of genomic intervals analyzed strongly affected the conclusions drawn--thus, answering the question "What genomic features affect integration?" requires carefully specifying the length scale of interest.

Binding Sites↗
PubMed1999

Progress in Targeted Gene Integration.

The ultimate goal of gene therapy is correction of the genetic disorders by introducing the genetic materials into targeted cells, and gene expression should be permanent and at an appropriate level. The widely used gene therapy vectors nowadays can not meet the demands. The targeted integration mediated by the homologous recombination is the best strategy for human gene therapy, because the gene expression is controlled by the natural elements. The progress in homologous recombination recently made it possible to correct the genetic disorder s by targeted integration of the interest gene.

Journal Article↗