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At least 217 records · Page 12Linked to original sources
PubMed2005

Targeted gene transfer to nigrostriatal neurons in the rat brain by helper virus-free HSV-1 vector particles that contain either a chimeric HSV-1 glycoprotein C-GDNF or a gC-BDNF protein.

Direct gene transfer into neurons has potential for both studying neuronal physiology and for developing gene therapy treatments for specific neurological conditions. Due to the heterogeneous cellular composition of the brain, cell-type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. The two prevalent approaches for achieving cell-type-specific expression are to use a cell-type-specific promoter to control recombinant gene expression or to modify a virus vector particle to target gene transfer to a specific cell type. Targeted gene transfer to multiple peripheral cell types has been described, but targeted gene transfer to a specific type of neuron in the brain has yet to be reported. Targeted gene transfer approaches with Herpes Simplex Virus (HSV-1) vectors have focused on modifying glycoprotein C (gC) to remove the heparin binding domain and add a binding activity for a specific protein on the cell surface. This study was designed to develop HSV-1 vectors that target gene transfer to cells that contain receptors for either glial-cell-line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF), such as nigrostriatal neurons. We isolated chimeric gC-GDNF or chimeric gC-BDNF constructs, and the resulting proteins were incorporated into HSV-1 virus particles. We performed helper virus-free HSV-1 vector packaging in the presence of each chimeric protein. The resulting vector stocks supported 2.2- to 5.0-fold targeted gene transfer to nigrostriatal neurons in the rat brain, compared to vector particles that contained wild-type (wt) gC. Gene transfer to nigrostriatal neurons by vector particles that contained chimeric gC-BDNF was reduced by preincubation with an anti-BDNF antibody. Targeted gene transfer to neurons that contain specific neurotrophic factor receptors may benefit specific physiological and gene therapy studies.

Animals↗
PubMed2006

Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells.

Gene targeting via homologous recombination is a powerful tool for studying gene function, but the targeting efficiency in human cell lines is too low for generating knockout mutants. Several cell lines null for the gene responsible for Bloom syndrome, BLM, have shown elevated targeting efficiencies. Therefore, we reasoned that gene targeting would be enhanced by transient suppression of BLM expression by RNA interference. To test this, we constructed a gene correction assay system to measure gene targeting frequencies using a disrupted hypoxanthine phosphoribosyltransferase (HPRT) locus in the human HT1080 cell line, and examined the effect of small interfering RNA (siRNA) for BLM on gene targeting. When HPRT-null cells pretreated with BLM siRNA were co-transfected with the siRNA and a gene correction vector, the gene targeting frequency was elevated three-fold, while the random integration frequency was marginally affected. Remarkably, in BLM heterozygous (+/-) cells derived from HPRT-null cells, the BLM siRNA treatment gave more than five-fold higher targeting frequencies, even with one-tenth the amount of BLM siRNA used for BLM+/+ cells. Furthermore, in the human pre-B cell line Nalm-6, the siRNA treatment enhanced gene targeting 6.3-fold and > 5.8-fold at the HPRT and adenine phosphoribosyltransferase (APRT) loci, respectively. These results indicate that transient suppression of BLM expression by siRNA stimulates gene targeting in human cells, facilitating a further improvement of gene targeting protocols for human cell lines.

Adenine Phosphoribosyltransferase↗
PubMed2002

Analysis of inhibitory action of modified U1 snRNAs on target gene expression: discrimination of two RNA targets differing by a 1 bp mismatch.

The modified U1 snRNA gene can suppress expression of a target transgene. In the present study, its potential utility to inhibit a dominant negative/gain of function mutation is explored. Using a green fluorescent protein (GFP) target gene, inhibition was achieved in all cells transduced with U1antiGFP directed at multiple sites within GFP. Using a chloramphenicol acetyltransferase (CAT) target gene, inhibition was not increased by increasing the hybridization domain from 10 to 16 bp or when a site in an upstream exon or intron was targeted. To determine if a U1 anti-target design could discriminate between two transcripts that differ by a 1-2 bp mismatch, GFPtpz and GFPsaph were chosen as targets because they share sequence homology except for three regions where a 1, 2 or 3 bp mismatch exists. The results demonstrated that U1antiGFP correctly reduced its cognate GFP expression by >90% and therefore U1 anti-target constructs are able to discriminate a 1 or 2 bp mismatch in their target mRNA. Thus, these U1 anti-target constructs may be effective in a strategy of somatic gene therapy for a dominant negative/gain of function mutation due to the discreteness of its discrimination. It may complement other anti-target strategies to reduce the cellular load of a mutant transcript.

3T3 Cells↗
PubMed2000

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium.

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Base Sequence↗
PubMed1995

Inactivation of Apoe and Apoc1 by two consecutive rounds of gene targeting: effects on mRNA expression levels of gene cluster members.

The genes encoding apolipoprotein (apo) E and apoC1 are, together with the gene for apoC2, located in a conserved gene cluster on human chromosome 19q12-13.2 and mouse chromosome 7. Although the significance of apoE as a ligand for receptor-mediated uptake of lipoprotein remnant particles is undisputed, the in vivo function of apoC1 and the possible interaction between apoE and apoC1 in the modulation of plasma cholesterol and triglyceride levels is far from understood. Our strategy to unravel the metabolic relationship between apoE and apoC1 in vivo is to first generate mice deficient in both apolipoproteins, enabling future production of transgenic mice with variable ratios of normal and mutant apoE and apoC1 on a null background. Here we report the creation and characterization of mice deficient in both apoE and apoC1. As these genes are tightly genetically linked, double-deficient mice were obtained by two consecutive rounds of gene targeting in mouse embryonic stem cells. Surprisingly, double inactivation of the Apoe and Apoc1 gene loci as well as single inactivations at either one of these loci were found to affect also the RNA expression levels of the other gene members in the Apoe-c1-c2 cluster. This indicates that targeted insertions are not necessarily neutral for the expression of nearby gene members in a given gene cluster. Homozygous Apoe-c1 knockout mice are hypercholesterolemic, with serum cholesterol levels of 12.5 +/- 4.3 mM compared with 2.9 +/- 0.5 mM in control mice, resembling mice solely deficient in apoE.

Animals↗
PubMed2002

New plasmid vectors for specific gene targeting in Spiroplasma citri.

In Spiroplasma citri gene inactivation through homologous recombination has been achieved by using the replicative, oriC plasmid pBOT1 as the disruption vector. However, plasmid recombination required extensive passaging of the transformants and, in most cases, recombination occurred at oriC rather than at the target gene. In the current study, we describe a new vector, in which the oriC fragment was reduced to the minimal sequences able to promote plasmid replication. Using this vector to inactivate the motility gene scm1 showed that size reduction of the oriC fragment did increase the frequency of recombination at the target gene. Furthermore, to avoid extensive passaging of the transformants, we developed a strategy in which the selective, tetracycline resistance phenotype can only be expressed once the plasmid has integrated into the chromosome by one single crossover recombination at the target gene. As an example, targeting of the spiralin gene is described.

Base Sequence↗
PubMed1991

Correction of a human beta S-globin gene by gene targeting.

As a step toward using gene targeting for gene therapy, we have corrected a human beta S-globin gene to the normal beta A allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the beta S-globin allele. A beta A-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total of approximately 29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the beta S allele to beta A was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5' to the human beta A-globin gene. Thus gene targeting can correct a beta S allele to beta A, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

Animals↗
PubMed1999

The molecular basis of multiple vector insertion by gene targeting in mammalian cells.

Gene targeting using sequence insertion vectors generally results in integration of one copy of the targeting vector generating a tandem duplication of the cognate chromosomal region of homology. However, occasionally the target locus is found to contain >1 copy of the integrated vector. The mechanism by which the latter recombinants arise is not known. In the present study, we investigated the molecular basis by which multiple vectors become integrated at the chromosomal immunoglobulin mu locus in a murine hybridoma. To accomplish this, specially designed insertion vectors were constructed that included six diagnostic restriction enzyme markers in the Cmu region of homology to the target chromosomal mu locus. This enabled contributions by the vector-borne and chromosomal Cmu sequences at the recombinant locus to be ascertained. Targeted recombinants were isolated and analyzed to determine the number of vector copies integrated at the chromosomal immunoglobulin mu locus. Targeted recombinants identified as bearing >1 copy of the integrated vector resulted from a Cmu triplication formed by two vector copies in tandem. Examination of the fate of the Cmu region markers suggested that this class of recombinant was generated predominantly, if not exclusively, by two targeted vector integration events, each involving insertion of a single copy of the vector. Both vector insertion events into the chromosomal mu locus were consistent with the double-strand-break repair mechanism of homologous recombination. We interpret our results, taken together, to mean that a proportion of recipient cells is in a predetermined state that is amenable to targeted but not random vector integration.

Animals↗
PubMed2005

A trial of somatic gene targeting in vivo with an adenovirus vector.

BACKGROUND: Gene targeting in vivo provides a potentially powerful method for gene analysis and gene therapy. In order to sensitively detect and accurately measure designed sequence changes, we have used a transgenic mouse system, MutaMouse, which has been developed for detection of mutation in vivo. It carries bacteriophage lambda genome with lacZ+ gene, whose change to lacZ-negative allele is detected after in vitro packaging into bacteriophage particles. We have also demonstrated that gene transfer with a replication-defective adenovirus vector can achieve efficient and accurate gene targeting in vitro. METHODS: An 8 kb long DNA corresponding to the bacteriophage lambda transgene with one of two lacZ-negative single-base-pair-substitution mutant allele was inserted into a replication-defective adenovirus vector. This recombinant adenovirus was injected to the transgenic mice via tail-vein. Twenty-four hours later, genomic DNA was extracted from the liver tissue and the lambda::lacZ were recovered by in vitro packaging. The lacZ-negative phage was detected as a plaque former on agar with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the lacZ-negative recombinant adenovirus injected mice was at the same level with the control mouse (approximately 1/10000). Our further restriction analysis did not detect any designed recombinant. CONCLUSION: The frequency of gene targeting in the mouse liver by these recombinant adenoviruses was shown to be less than 1/20000 in our assay. However, these results will aid the development of a sensitive, reliable and PCR-independent assay for gene targeting in vivo mediated by virus vectors and other means.

Journal Article↗
PubMed1994

Gene targeting and synaptic plasticity.

Gene targeting is revealing new molecular functions by creating very specific developmental, physiological and behavioral perturbations, and providing new insights into biochemical pathways underlying synaptic plasticity. Recent studies of mice carrying mutations in genes thought to be involved in modulating synaptic transmission have been subject to integrated biochemical, physiological and behavioral analyses.

Animals↗
PubMed1997

ETS target genes: identification of egr1 as a target by RNA differential display and whole genome PCR techniques.

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing's sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

3T3 Cells↗
PubMedDate not supplied

Functions of mammalian Smad genes as revealed by targeted gene disruption in mice.

The Smad genes are the intracellular mediators of TGF-beta signals. Targeted mutagenesis in mice has yielded valuable new insights into the functions of this important gene family. These experiments have shown that Smad2 and Smad4 are needed for gastrulation, Smad5 for angiogenesis, and Smad3 for establishment of the mucosal immune response and proper development of the skeleton. In addition, these experiments have shown us the importance of gene dosage in this family, as several of its members yielded haploinsufficiency phenotypes. These include gastrulation and craniofacial defects for Smad2, accelerated wound healing for Smad3, and the incidence of gastric cancer for Smad4. Combinatorial genetics has also revealed functions of Smads in left/right isomerism and liver development.

Animals↗
PubMed2006

A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs.

Alternaria brassicicola causes black spot disease of cultivated Brassicas and has been used consistently as a necrotrophic fungal pathogen for studies with Arabidopsis. In A. brassicicola, mutant generation has been the most rate-limiting step for the functional analysis of individual genes due to low efficiency of both transformation and targeted integration. To improve the targeted gene disruption efficiency as well as to expedite gene disruption construct production, we used a short linear construct with minimal elements, an antibiotic resistance selectable marker gene, and a 250- to 600-bp-long partial target gene. The linear minimal element (LME) constructs consistently produced stable transformants for diverse categories of genes. Typically, 100% of the transformants were targeted gene disruption mutants when using the LME constructs, compared with inconsistent transformation and usually less than 10% targeted gene disruption with circular plasmid disruption constructs. Each mutant displayed a unique molecular signature thought to originate from endogenous exonuclease activities in fungal cells. Our data suggests that a DNA double-stranded break repair mechanism (DSBR) functions to increase targeting efficiency. This method is advantageous for high throughput gene disruption, overexpression, and reporter gene introduction within target genes, especially for asexual filamentous fungi where genetic approaches are unfavorable.

Acetylglucosaminidase↗
PubMed2004

Unexpected embryonic stem (ES) cell mutations represent a concern in gene targeting: lessons from "fierce" mice.

The exceptional value of gene targeting technology to generate mouse models of human disease exists under the shadow of potential genetic errors. We previously observed an unexpected brain-behavior phenotype that resulted from a gene-targeting experiment designed to delete the Zfa gene. Given that the transcription of Zfa is restricted to the germ cell lineage of adult testis, it was both a surprise and a concern when the resulting mice had a phenotype present in both sexes that included abnormal brains and violent behavior. We hypothesized that an unrelated mutation may have been responsible for the unexpected phenotype. Here we show that the single gene mutation, Nr2e1(frc) (fierce), which was responsible for the brain-behavior phenotype, existed in the embryonic stem (ES) cell even before the derivation of the Zfa knockout mice. Our work thus highlights a concern in gene targeting, namely, that ES cells can harbor unexpected mutations, which can lead to genotype-phenotype misattribution. Based on our findings, we caution the gene-targeting community to use low-passage ES cells, to characterize mice derived from more than one independently targeted ES cell clone, and to backcross mice to allow for segregation of distant but linked mutations.

Amino Acid Sequence↗
PubMedDate not supplied

Identification and validation of candidate Myb target genes.

While a considerable number of candidate Myb target genes have been reported to date, most of these are likely to play little or no role in transformation by myb oncogenes. Here we have used a conditionally myb-transformed myeloid cell line (ERMYB) to further examine Myb regulation of one candidate target gene--c-myc--that has the potential to affect cell proliferation. It was found that the major influence on c-myc expression was the presence of cytokine (GM-CSF) rather than Myb activity. We also describe the application of PCR-based subtractive hybridization and low-density cDNA array screening, in conjunction with the ERMYB line, to the identification of additional Myb target genes. Preliminary identification of a number of candidates is reported; these include myeloperoxidase, which is known to have essential Myb-binding sites in its regulatory region.

Cell Line, Transformed↗
PubMed1996

An element in the endogenous IgH locus stimulates gene targeting in hybridoma cells.

Gene targeting of the immunoglobulin (Ig) heavy chain locus is the basis of improved methods of investigating gene expression and of antibody engineering. The VH-Cmu intron is a convenient region for mediating homologous recombination events which result in production of Ig bearing an altered heavy chain. Also, this segment includes several elements which are important for gene expression, replication and isotype switching: in some cases it will be advantageous to alter these processes by modifying this intron. Considering that multiple targeting steps might be needed to accomplish all the requisite changes, it is important to know whether any of the anticipated modifications also alter the recombinogenicity of the IgH locus. To test this possibility we have measured the frequency at which a mutation in the Cmu3 exon of the endogenous mu gene is corrected by homologous recombination with a transfected segment of Cmu DNA. Comparison of recombination frequencies in several engineered hybridomas indicates that deletion of a 7.1 kb segment from the VH-Cmu intron depresses recombination by approximately 10-fold.

Base Sequence↗
PubMed2004

Oligonucleotide mediated gene targeting in mammalian cells.

Gene targeting can be loosely defined as a process through which a specific chromosomal sequence is recognized and bound by a reagent designed for the purpose. The endpoint may be modulation of events at the target, such as transcription, or a permanent change in sequence at the site. A facile strategy for mammalian cells would have broad applications in basic and applied research, including gene therapy. Although approaches based on homologous recombination are routinely employed for transgenic animal construction, they are too laborious and inefficient for broader use. Consequently there has been a longstanding interest in developing effective synthetic reagents. Sequence recognition can be either at the level of a single strand or via the major or minor grooves, and specific approaches for each route are under development. In this review several oligonucleotide-based strategies will be discussed. These include single and double strand oligonucleotides designed to attack single strand targets, and triple helix forming olgonucleotides and peptide nucleic acids, intended for double stranded targets.

Animals↗