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Results for “targeted integration”

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At least 145 records · Page 8Linked to original sources
PubMed1992

Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.

We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.

Blotting, Southern↗
PubMed1996

Hepatitis B virus and hepatocellular carcinoma.

Hepatitis B virus (HBV) has been implicated as a causative factor for the development of hepatocellular carcinoma (HCC). However, no oncogenes have been identified within the HBV genome and furthermore, the virus is frequently fragmented after it integrates into the hepatocyte genome. Therefore it has been difficult to understand how HBV brings about genetic damage to result in HCC. Recent data from our laboratory show that despite fragmentation, the HBx gene is consistently retained in a functional form and therefore suggest that this gene is important to HCC development. In 4 out of 6 HCC cell lines studied, the DR2 integration sequence was destroyed during integration and points to a possible preferred target integration site. The growing importance of HBx in HCC development is supported by data from other laboratories showing that transgenic mice carrying the HBx gene alone frequently develop HCC. This review discusses the role of HBV and other factors involved in liver carcinogenesis.

Animals↗
PubMed1991

Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae.

Targeted integration of the yeast plasmid pMIRY2 into the ribosomal DNA (rDNA) of Saccharomyces cerevisiae by homologous recombination results in transformants carrying 100-200 copies of the plasmid per cell which are stably maintained over a large number of generations [Lopes et al., Gene 79 (1989) 199-206]. These properties make pMIRY2 an attractive vector for high-level production of (heterologous) proteins by yeast cells. We have investigated the mechanism underlying high-copy-number (hcn) integration of pMIRY-type plasmids and show that either targeting to a location outside the rDNA locus or use of the wild-type LEU2, instead of the deficient LEU2d gene, as selection marker reduces the copy number to the low value characteristic of standard integrating (YIp-type) yeast plasmids. Further experiments demonstrate that the hcn of pMIRY-type plasmids is achieved by amplification of a small number of copies initially integrated into the rDNA locus. Amplification depends upon the strong selection pressure created by the extremely low expression of the deficient LEU2d gene, but not on the presence of this gene per se. The hcn integration also occurs when either the TRP1 or URA3 gene is used as the selection marker, provided expression of the marker gene is severely curtailed, e.g., by removal of most of its 5'-flanking region.

DNA, Fungal↗
PubMed1998

Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre.

The Cre/lox system was used to obtain targeted integration of an Agrobacterium T-DNA at a lox site in the genome of Arabidopsis thaliana. Site-specific recombinants, and not random events, were preferentially selected by activation of a silent lox-neomycin phosphotransferase (nptII) target gene. To analyse the effectiveness of Agrobacterium-mediated transfer we used T-DNA vectors harbouring a single lox sequence (this vector had to circularize at the T-DNA left- and right-border sequences prior to site-specific integration) or two lox sequences (this vector allowed circularization at the lox sequences within the T-DNA either prior to or after random integration, followed by targeting of the circularized vector), respectively. Furthermore, to control the reversibility of the integration reaction, Cre recombinase was provided transiently by using a cotransformation approach. One precise stable integrant was found amongst the recombinant calli obtained after transformation with a double-lox T-DNA vector. The results indicate that Agrobacterium-mediated transformation can be used as a tool to obtain site-specific integration.

Arabidopsis↗
PubMed1992

Retinal ganglion cell degeneration following loss of postsynaptic target neurons in the dorsal lateral geniculate nucleus of the adult cat.

Kainic acid was used to produce selective degeneration of neurons in the dorsal lateral geniculate nucleus of the adult cat. This degeneration mimics the rapid loss of geniculate neurons seen after visual cortex ablation in the neonate. Following survivals of 2, 4, or 6 months, the geniculate was injected with horseradish peroxidase and the retinae were examined for the presence of retrogradely labeled cells. Analysis of ganglion cell density in peripheral nasal retina revealed a 58% loss of cells overall at 6 months. The proportion of cells labeled with horseradish peroxidase decreased more rapidly, until none were labeled at 6 months. Separate analysis of small, medium, and large ganglion cell populations revealed that only medium-sized cells were lost at 2 months whereas both medium and large cells were lost at 4 and 6 months. By 6 months, 92% of medium cells and 65% of large cells had degenerated. These results show that mature retinal ganglion cells in the cat maintain a dependence on target integrity for their continued survival. When the appropriate target is lost, the ganglion cells respond first by axon terminal retraction and then by cell death.

Analysis of Variance↗
PubMed1992

Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination.

Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.

Blotting, Northern↗
PubMed2001

Yeast vectors for integration at the HO locus.

We have constructed new yeast vectors for targeted integration of desired sequences at the Saccharomyces cerevisiae HO locus. Insertion at HO has been shown to have no effect on yeast growth, and thus these integrations should be neutral. One vector contains the KanMX selectable marker, and integrants can be selected by resistance to G418. The other vector contains the hisG-URA3-hisG cassette, and integrants can be selected by uracil prototrophy. Subsequent growth on 5-FOA permits identification of colonies where recombination between the hisG tandem repeats has led to loss of the URA3 marker and return to uracil auxotrophy. We also describe several new bacterial polylinker vectors derived from pUC21 (ampicillin resistance) and pUK21 (kanamycin resistance).

Ampicillin Resistance↗
PubMed1992

Analysis of sequence specificity of 5-bromodeoxyuridine-induced reversion in cells containing multiple copies of a mutant gpt gene.

For studies on molecular mechanisms of mutagenesis, it would be advantageous to transfer mutant genes with specific alterations into mammalian cells and use the transformed cells in reversion analyses. In the present paper, we describe an efficient method for analyzing reversion events occurring in cells that possess multiple copies of a mutational target gene. This method involves amplification of the chromosomally integrated target genes with the polymerase chain reaction (PCR) and restriction endonuclease digestion of the amplified product. Single reversion events that either create or destroy restriction endonuclease recognition sequences that encompass the site of the original mutation can be identified in a background of 10-20 copies of the gene that retain the mutant sequence. Using this method, we have analyzed revertants induced by 5-bromodeoxyuridine (BrdU) in a Chinese hamster ovary cell line that possesses multiple copies of a mutant bacterial gpt gene containing a specific alteration. The results of this study not only demonstrate the effectiveness of this method for analyzing reversion of a single gene copy in transfectants possessing multiple copies of a mutant target gene, but also demonstrate that the sequence specificity for BrdU-induced mutations is the same in Chinese hamster cells as previously observed with mouse cells.

Animals↗
PubMed1993

Human immunodeficiency virus type 1 DNA integration: fine structure target analysis using synthetic oligonucleotides.

The target specificity of DNA strand transfer mediated by human immunodeficiency virus type 1 integrase was examined in vitro with synthetic oligonucleotides. Although insertion occurred at most locations in the target, some sites were preferred over others by at least 15-fold. Changing the nucleotide sequence of the target changed the distribution of preferred sites in complex ways, some of which included changes in target preference distant from the sequence alteration. Alignment of target sequences revealed that adenosine is preferred adjacent to the insertion site. Strand transfer occurred to within 2 nucleotides of the 3' end and to within 3 nucleotides of the 5' end of the target. This suggests that only 2 or 3 nucleotides flanking the target site are required for integration; such restricted contact with target DNA would allow integrase to insert the two ends of viral DNA into two closely spaced sites in host DNA, consistent with the concerted in vivo integration reaction that generates a 5-bp target duplication.

Base Sequence↗
PubMed2000

[Effects of gene copy number and chromosomal position on the expression of a modified HBsAg gene SA-28 in yeast].

The effect of gene copy number and chromosomal position on heterologous gene expression in Saccharomyces cerevisae has been tentatively investigated using a modified hepatitis B virus surface antigen SA-28 gene. The plasmids containing different copies of SA-28 gene expression cassette were integrated into different chromosomal loci via FLP recombinase mediated targeted integration. The expression of SA-28 gene in the resultant transformants was then measured. The result indicated that the expression of SA-28 gene was positively related to the gene dosage at HIS3 locus and had chromosomal polarity at several chromosomal loci.

Chromosome Mapping↗
PubMed2001

HIV-1 preintegration complexes preferentially integrate into longer target DNA molecules in solution as detected by a sensitive, polymerase chain reaction-based integration assay.

After entering a cell and undergoing reverse transcription, the retroviral genome is contained in a preintegration complex (PIC) that mediates its integration into host cell DNA. PICs have been shown to prefer torsionally strained DNA, but the effect of target DNA length has not been previously examined. In this report, concatemerization of a repeating 105-base pair unit was used to vary target DNA length independently from basic DNA sequence, while maintaining both PICs and target DNAs in solution. Integration junctions were quantified by real-time fluorescence-monitored polymerase chain reaction amplification using primers in the viral long terminal repeat and the target DNA. Unreacted target DNA severely inhibited the post-reaction polymerase chain reaction detection step, requiring its removal using lambda exonuclease digestion. Integration into a 32-unit concatemer of target DNA was markedly more efficient than integration into a monomeric unit, indicating that longer target DNA was preferred. This substrate was used to construct a simple, robust, and adaptable assay that can serve as a method for studying the host cell factors that enhance PIC integration, and as a drug discovery platform for integration inhibitors active against PICs.

Binding, Competitive↗
PubMed2026

Engineered genomic attachment sites for site-specific recombinases enable high-efficiency integration in plants and human cells.

Here we developed a DNA-centric strategy for optimizing site-specific recombination by rationally engineering chimeric attachment sites. The high-activity att variants enhance Bxb1-mediated integration efficiency in human cells and plants. Among these att variants, the engineered attB(V111) site achieved 51.9% integration efficiency in HEK293T cells (1.7-fold versus wild-type attB) and 35.6% in rice protoplasts (4.4-fold versus wild-type attB). When paired with an engineered single protein mutant in the Bxb1 catalytic domain, the optimized system achieved targeted integration efficiencies of 31% for a CD19 chimeric antigen receptor cassette and 25% for an ornithine transcarbamylase expression cassette in human cells. In rice, these engineered variants enabled integration of a 5.8 kb herbicide-resistance cassette at a targeted genomic locus, with stable integration detected in 24% of regenerated plants. Oxford Nanopore-based long-read sequencing of edited plants reveals complete and precise insertion with high specificity. Propagation of edited seedlings to T1 plants confirms heritable editing to future generations. This approach provides a safe, broadly applicable approach for recombinase-based genome editing.

Journal Article↗
PubMed2000

A large database of chicken bursal ESTs as a resource for the analysis of vertebrate gene function.

Chicken B cells create their immunoglobulin repertoire within the Bursa of Fabricius by gene conversion. The high homologous recombination activity is shared by the bursal B-cell-derived DT40 cell line, which integrates transfected DNA constructs at high rates into its endogenous loci. Targeted integration in DT40 is used frequently to analyze the function of genes by gene disruption. In this paper, we describe a large database of >7000 expressed sequence tags (ESTs) from bursal lymphocytes that should be a valuable resource for the identification of gene disruption targets in DT40. ESTs of interest can be recognized easily by online or keyword searches. Because the database reflects the gene expression profile of bursal lymphocytes, it provides valuable hints as to which genes might be involved in B-cell-specific processes related to immunoglobulin repertoire formation, signal transduction, transcription, and apoptosis. This large collection of chicken ESTs will also be useful for gene expression studies and comparative gene mapping within the chicken genome project. Details of the bursal EST sequencing project and access to database search forms can be found on the DT40 web site (http://genetics.hpi.uni-hamburg.de/dt40.html).

Animals↗
PubMed1995

Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element.

The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

DNA Nucleotidyltransferases↗
PubMed1991

Gene targeting in Chinese hamster ovary cells is conservative.

Two fundamentally different pathways for homologous recombination have been identified in mammalian cells. For most chromosomal recombination events, two copies of a homologous sequence recombine to yield two copies in the products; such events are said to be conservative because the number of copies is preserved. By contrast, virtually all extrachromosomal recombination events are nonconservative; two copies recombine to give a product containing a single intact copy (the other copy is destroyed in the mechanism). Since gene targeting involves an introduced (extrachromosomal) plasmid and a chromosomal target, it was not clear which pathway would apply. We used a marked vector to determine whether targeted integrants were products of recombination events that involved two copies (the conservative pathway) or three copies (the nonconservative pathway) of the homologous sequence. Among 51 gene targeting events, we identified 17 homologous integrants and analyzed their structures. All match the predictions for a conservative pathway. We conclude that the principal pathway for gene targeting in mammalian cells is conservative.

Adenine Phosphoribosyltransferase↗
PubMed2005

Differential chromosome control of ploidy in the yeast Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, aneuploidy is well tolerated and stable. We analysed whether the induced loss of a disomic chromosome favours endo-reduplication of the remaining chromosome or the cells prefer to retain the acquired euploidy. Chromosome VIII disomes and trisomes were tagged with GFP (green fluorescent protein), DsRed (red fluorescent protein) and BFP (blue fluorescent protein) integrated at the thr1 locus, using our newly designed STIK (specific targeted integration of kanamycin resistance-associated, non-selectable DNA) plasmid system. A knockout cassette for centromere 8 was constructed with the hygromycin-B marker, which was transformed into the strains. The transformants lost sensitivity to hygromycin, thereby indicating the event of centromere replacement. Quantitative PCR and Southern analysis were performed for chromosome VIII copy number determination by probing the markers located on both the right (ARG4 and THR1) and left (GUT1) arm whereas, for chromosome V, markers such as HIS1, located on right arm, and URA3, on left arm, were used. The loss of an extranumerary chromosome VIII in a disome and trisome leads to stable euploidy. Furthermore, in a wild-type diploid, deletion of a copy of chromosome VIII, leads to monosomy, and restoration of euploidy after 22 generations, by reduplication of chromosome VIII, and consequent loss of heterozygosis (LOH). However, chromosome V knockouts in chromosome VIII trisome, still showed LOH and duplication of chromosome V, with return to the original aneuploid condition. These results suggest that yeast cells could control the integrity of their genetic complement acting at the individual chromosome level.

Aneuploidy↗
PubMed2006

Retroviral DNA integration: HIV and the role of LEDGF/p75.

To replicate, a retrovirus must integrate a DNA copy of its RNA genome into a chromosome of the host cell. Integration is not random in the host genome but favors particular regions, and preferences differ among retroviruses. Several mechanisms might play a part in this favored integration targeting: (i) open chromatin might be preferentially accessible for viral DNA integration; (ii) DNA replication during cell division might facilitate access of integration complexes to favored sites; and (iii) cellular proteins bound to the host chromosome might tether integration complexes to favored regions. This review summarizes recent advances in understanding the mechanisms of retroviral integration, focusing on LEDGF/p75--the first cellular protein shown to have a role in directing HIV DNA integration. Studies on LEDGF/p75 indicate that it directs HIV integration site selection by a tethering interaction, whereas the chromatin accessibility or cell cycle models are less well supported. Understanding viral integration will help improve the safety of retrovirus-based vectors used in gene therapy.

Chromatin↗
PubMed1987

Modification of DNA ends can decrease end joining relative to homologous recombination in mammalian cells.

In animal cells, exogenous DNA recombines into random chromosomal sites much more frequently than it recombines into homologous sites. Free DNA ends are "recombinogenic" in both processes. To test the effects of specific ends on analogous extrachromosomal processes, we constructed a linear genome of simian virus 40 with terminal repeated sequences. After transfection into monkey cells, the model substrate can circularize by end joining (analogous to random integration) or by homologous recombination between its terminal repeats (analogous to targeted recombination). Since the two types of recombination are in competition with one another, the ratio of homologous-recombination to end-join products is a sensitive indicator of the differential effects of specific ends. Substrates with blunt ends, complementary sticky ends, or mismatched ends generated the same ratio of homologous-recombination to end-join products. However, addition of dideoxynucleotides to the 3' hydroxyls of the substrate decreased the frequency of end joining by a factor of 5-6 relative to homologous recombination. Thus, the frequency of end joining can be decreased relative to that of homologous recombination by modification of the ends of the input DNA. These results suggest an approach to altering the ratio of random to targeted integration in mammalian cells.

Animals↗