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At least 613 records · Page 34Linked to original sources
PubMed1998

Human gene targeting by viral vectors.

Stable transduction of mammalian cells typically involves random integration of viral vectors by non-homologous recombination. Here we report that vectors based on adeno-associated virus (AAV) can efficiently modify homologous human chromosomal target sequences. Both integrated neomycin phosphotransferase genes and the hypoxanthine phosphoribosyltransferase gene were targeted by AAV vectors. Site-specific genetic modifications could be introduced into approximately 1% of cells, with the highest targeting rates occurring in normal human fibroblasts. These results suggest that AAV vectors could be used to introduce specific genetic changes into the genomic DNA of a wide variety of mammalian cells, including therapeutic gene targeting applications.

Adenoviridae↗
PubMed2005

Instability of chimaeric antibody secretion by anti-carcinoembryonic antigen producing hybridoma cells after gene targeting.

OBJECTIVE: To produce a chimaeric version of the 11-285-14 anti-carcinoembryonic antigen (CEA) monoclonal antibody using a gene targeting approach. MATERIALS AND METHODS: A replacement vector was constructed to insert the human constant gamma1 gene within the mouse heavy chain locus of 11-285-14 hybridoma cells. The mouse constant gamma1 gene (1.5 kb) and the mouse mu intron fragment (2.2 kb) were amplified by PCR and cloned into a pKO Scrambler vector. The human constant gamma1 gene fragment (2.2 kb) was cloned next to the intron fragment. Resistant colonies were screened by ELISA for the presence of the human isotype in their supernatants. RESULTS: Of the 4,370 resistant colonies obtained, 87 colonies showed secretion of the human isotype at levels between 4 and 32 ng/ml. PCR and Southern blot results confirmed the correct integration of the human gene by homologous recombination within the heavy chain locus. Most of the producers ceased to express the human isotype within a few weeks after the initial positive ELISA results. Instability of secretion could not be explained by genetic instability in all the clones, which suggests the presence of other undefined epigenetic or physiologic mechanisms. CONCLUSION: Gene targeting resulted in transformants with unstable and low production rates of chimaeric anti-CEA antibody.

Alleles↗
PubMed1997

[Inhibition of cell growth and target gene expression of human pancreatic carcinoma cells by modified antisense oligodeoxynucleotide].

OBJECTIVE: To investigate the inhibitory effects of modified antisense oligodeoxynucleotide on cell growth, 3H-TdR incorporation rate and target gene expressions of human pancreatic carcinoma cells as a comparison with the effectiveness of nonmodified antisense oligodeoxynucleotide (ASODN) resported previously. METHODS: Synthesized modified antigsense oligodeoxynucleotides (antisense phosphorothioate oligodeoxynucleotides, ASPODN) complementary to the cap regions of c-myc and Ki-ras genes were used to treat PC-2 and PC-3 human pancreatic carcinoma cell line cells with multiple small (10 micrograms) doses or one single dose (15 micrograms). After treatment, cell growth rates and 3H-TdR incorporation rates were estimated, and the concurrent oncogene expressions were studied by adopting RT-PCR technique. RESULTS: After multiple ASPODN exposures, the cell growth rates and 3H-TdR incorporation rates were significantly inhibited, the inhibition was maintained for more than two weeks. On the 14th day, the cell growth rates of the ASPODN groups were reduced to 38%-43% of that of the controls, and the 3H-TdR incorporation rates were 18%-33% of that of the controls, there were also marked inhibition or down-regulation of target genes (c-myc and Ki-ras) expressions. After the 15 micrograms single dose treatment, the inhibition of cell growth, 3H-TdR incorporation and target gene expressions lasted for 4 days. CONCLUSIONS: The results of this study confirm the fact that ASPODN exerts a more strong inhibitory effect on pancreatic carcinoma cells than the non-modified ASODN.

Adenocarcinoma↗
PubMed2001

Polyethylenimine/DNA complexes shielded by transferrin target gene expression to tumors after systemic application.

Systemic application of positively charged polycation/DNA complexes has been shown to result in predominant gene expression in the lungs. Targeting gene expression to other sites, eg distant tumors, is hampered by nonspecific interactions largely due to the positive surface charge of transfection complexes. In the present study we show that the positive surface charge of PEI (25 kDa branched or 22 kDa linear)/DNA complexes can be efficiently shielded by covalently incorporating transferrin at sufficiently high densities in the complex, resulting in a dramatic decrease in nonspecific interactions, eg with erythrocytes, and decreased gene expression in the lung. Systemic application of transferrin-shielded PEI/DNA complexes into A/J mice bearing subcutaneously growing Neuro2a tumors via the tail vein resulted in preferential (100- to 500-fold higher) luciferase reporter gene expression in distant tumors as compared with the major organs including the lungs. Tumor targeting is also demonstrated by DNA uptake and beta-galactosidase gene expression in tumor cells. Assessing DNA distribution following systemic application significant amounts of DNA were found in the liver and tumor. However, in the liver, DNA was mainly taken up by Kupffer cells and degraded without significant transgene expression. In the tumor, DNA was associated mainly with tumor cells and frequently found near structures which resemble primitive blood vessels.

Animals↗
PubMed2002

Identification of cytokine-induced nuclear factor-kappaB target genes in ovarian and breast cancer cells.

NF-kappaB is a pleiotropic transcription factor controlling the expression of many genes and viruses. NF-kappaB plays a role in immune response, cellular adhesion or acute phase response. It also inhibits apoptosis and favors cancer cell survival. We studied the expression of genes controlled by NF-kappaB in ovarian and breast adenocarcinoma cancer cells. We stably transfected OVCAR-3 and MCF7 A/Z cells with an expression vector coding for the mutated inhibitor IkappaBalpha, which sequesters NF-kappaB in the cytoplasm. We stimulated control and IkappaBalpha expressing cells with IL-1beta or TNF-alpha and extracted the RNA, which was reverse-transcribed and hybridized to DNA microarrays. Several of the genes identified were not known as NF-kappaB target genes. Among them, we confirmed the differential expression of ephrin-A1 and caveolin-1 by quantitative real-time polymerase chain reaction. Our results showed an NF-kappaB-dependent induction of ephrin-A1 and caveolin-1 mRNAs after stimulation with TNF-alpha and IL-1beta, confirming that NF-kappaB controls target genes implied in tumor angiogenesis and cell transformation.

Breast Neoplasms↗
PubMed2006

Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth.

ALR (MLL2) is a member of the human MLL family, which belongs to a larger SET1 family of histone methyltransferases. We found that ALR is present within a stable multiprotein complex containing a cohort of proteins shared with other SET1 family complexes and several unique components, such as PTIP and the jumonji family member UTX. Like other complexes formed by SET1 family members, the ALR complex exhibited strong H3K4 methyltransferase activity, conferred by the ALR SET domain. By generating ALR knockdown cell lines and comparing their expression profiles to that of control cells, we identified a set of genes whose expression is activated by ALR. Some of these genes were identified by chromatin immunoprecipitation as direct ALR targets. The ALR complex was found to associate in an ALR-dependent fashion with promoters and transcription initiation sites of target genes and to induce H3K4 trimethylation. The most characteristic features of the ALR knockdown cells were changes in the dynamics and mode of cell spreading/polarization, reduced migration capacity, impaired anchorage-dependent and -independent growth, and decreased tumorigenicity in mice. Taken together, our results suggest that ALR is a transcriptional activator that induces the transcription of target genes by covalent histone modification. ALR appears to be involved in the regulation of adhesion-related cytoskeletal events, which might affect cell growth and survival.

Animals↗
PubMed1999

Novel strategy yields candidate Gsh-1 homeobox gene targets using hypothalamus progenitor cell lines.

We describe the successful application of a strategy that potentially provides for an efficient and universal screen for downstream gene targets. We used the promoter of the Gsh-1 homeobox gene to drive expression of the SV40 T-antigen gene in transgenic mice. We have previously shown that the Gsh-1 homeobox gene is expressed in discrete domains of the ganglionic eminences, diencephalon, and hindbrain during brain development. Gsh-1-SV40 T transgenic mice showed cellular hyperplasia in regions of the brain coincident with Gsh-1 expression. The Gsh-1-SV40 T transgene was introduced, by breeding, into Gsh-1 homozygous mutant mice, and Gsh-1 -/- cell lines were made. Clonal cell lines were generated and analyzed by Northern blot hybridizations and Affymetrix GeneChip probe arrays to determine gene expression profiles. The results indicate that the cell lines remain representative of early developmental stages. Further, immunocytochemistry showed uniformly high levels of nestin expression, typical of central nervous system progenitor cells, and the absence of terminal differentiation markers of neuronal cells. One clonal cell line, No. 14, was then stably transfected with a tet-inducible Gsh-1 expression construct and subcloned. The starting clone 14, together with the uninduced and induced subclones, provided cell populations with varying levels of Gsh-1 expression. Differential display and Affymetrix GeneChip probe arrays were then used to identify transcript differences that represent candidate Gsh-1 target genes. Of particular interest, the drm and gas1 genes, which repress cell proliferation, were observed to be activated in Gsh-1-expressing cells. These observations support models predicting that homeobox genes function in the regulation of cell proliferation.

Animals↗
PubMed2005

Specific gene targeting in Spiroplasma citri: improved vectors and production of unmarked mutations using site-specific recombination.

In Spiroplasma citri, where homologous recombination is inefficient, specific gene targeting could only be achieved by using replicative, oriC plasmids. To improve the probability of selecting rare recombination events without fastidious, extensive passaging of the transformants, a new targeting vector was constructed, which was used to inactivate the crr gene encoding the IIA component of the glucose phosphotransferase system (PTS) permease. Selection of recombinants was based on a two-step strategy using two distinct selection markers, one of which could only be expressed once recombination had occurred through one single crossover at the target gene. According to this strategy, spiroplasmal transformants were screened and multiplied in the presence of gentamicin before the crr recombinants were selected for their resistance to tetracycline. In contrast to the wild-type strain GII-3, the crr-disrupted mutant GII3-gt1 used neither glucose nor trehalose, indicating that in S. citri the glucose and trehalose PTS permeases function with a single IIA component. In addition, the feasibility of using the transposon gammadelta TnpR/res recombination system to produce unmarked mutations in S. citri was demonstrated. In an arginine deiminase (arcA-disrupted) mutant, the tetM gene flanked by the res sequences was efficiently excised from the chromosome through expression of the TnpR resolvase from a replicative oriC plasmid. Due to oriC incompatibility, plasmid loss occurred spontaneously when selection pressure was removed. This approach will be helpful for constructing unmarked mutations and generating multiple mutants with the same selection marker in S. citri. It should also be relevant to other species of mollicutes.

DNA, Bacterial↗
PubMed2006

In vivo detection of Egr2 binding to target genes during peripheral nerve myelination.

Egr2/Krox20 is a zinc finger transactivator that regulates a diverse array of genes required for peripheral nerve myelination. Although several studies have elucidated the Egr2-regulated gene network, it is not clear if Egr2 regulates its target genes directly or indirectly through induction of other transactivators. Moreover, very few Egr2 binding sites have been identified in regulatory elements of myelin genes. To address this issue, we have successfully adapted chromatin immunoprecipitation assays to test if Egr2 binds directly to target genes in myelinating rat sciatic nerve. These experiments demonstrate direct binding of Egr2 to previously described binding sites within the Schwann cell enhancer of the myelin basic protein gene. Furthermore, we show Egr2 binding to a conserved site within the myelin-associated glycoprotein gene. Finally, our experiments provide the first evidence that Egr2 directly regulates expression of desert hedgehog, which is critically involved in development, maintenance and regeneration of multiple nerve elements including myelinated fibers. Surprisingly, this analysis has identified an apparent preponderance of Egr2 binding sites within conserved intron sequences of several myelin genes. Application of chromatin immunoprecipitation analysis to myelination in vivo will prove to be a valuable asset in assaying transcription factor binding and chromatin modifications during activation of myelin genes.

Animals↗
PubMed2005

pRb-Independent growth arrest and transcriptional regulation of E2F target genes.

The retinoblastoma tumor suppressor (pRb) has traditionally been studied as a negative regulator of cell cycle progression through its interactions with the E2F family of transcription factors. Utilizing prostate epithelial cell lines established from Rb+/+ and Rb-/- prostate tissues, we previously demonstrated that Rb-/- epithelial cells were not transformed and retained the ability to differentiate in vivo despite the lack of pRb. To further study the effects of pRb loss in an epithelial cell population, we utilized oligonucleotide microarrays to identify any pRb-dependent transcriptional regulation during serum depletion-induced growth arrest. These studies identified 120 unique transcripts regulated by growth arrest in Rb+/+ cells. In these wild-type cells, the majority (80%) of altered transcripts were downregulated, including 40 previously identified E2F target genes. Although the transcriptional repression of E2F target genes is characteristic of pRb pocket protein family activity, further analysis revealed that, compared to Rb+/+ cells, Rb-/- cells exhibited a nearly identical response for all transcripts including those of E2F target genes. These findings demonstrate that pRb is not strictly required for the vast majority of transcriptional alterations associated with growth arrest.

Animals↗
PubMed2004

Lymphotoxin and LIGHT signaling pathways and target genes.

Lymphotoxins (LT alpha and LT beta), LIGHT [homologous to LT, inducible expression, competes with herpes simplex virus (HSV) glycoprotein D for HSV entry mediator (HVEM), a receptor expressed on T lymphocytes], tumor necrosis factor (TNF), and their specific receptors LT beta R, HVEM, and TNF receptor 1 (TNFR1) and TNFR2, form the immediate family of the larger TNF superfamily. These cytokines establish a critical communication system required for the development of secondary lymphoid tissues; however, knowledge of the target genes activated by these signaling pathways is limited. Target genes regulated by the LT alpha beta-LT beta R pathway include the tissue-organizing chemokines, CXCL13, CCL19, and CCL21, which establish cytokine circuits that regulate LT expression on lymphocytes, leading to organized lymphoid tissue. Infectious disease models have revealed that LT alpha beta pathways are also important for innate and adaptive immune responses involved in host defense. Here, regulation of interferon-beta by LT beta R and TNFR signaling may play a crucial role in certain viral infections. Regulation of autoimmune regulator in the thymus via LT beta R implicates LT/LIGHT involvement in central tolerance. Dysregulated expression of LIGHT overrides peripheral tolerance leading to T-cell-driven autoimmune disease. Blockade of TNF/LT/LIGHT pathways as an intervention in controlling autoimmune diseases is attractive, but such therapy may have risks. Thus, identifying and understanding the target genes may offer an opportunity to fine-tune inhibitory interventions.

Animals↗
PubMed2004

Wnt target genes identified by DNA microarrays in immature CD34+ thymocytes regulate proliferation and cell adhesion.

The thymus is seeded by very small numbers of progenitor cells that undergo massive proliferation before differentiation and rearrangement of TCR genes occurs. Various signals mediate proliferation and differentiation of these cells, including Wnt signals. Wnt signals induce the interaction of the cytoplasmic cofactor beta-catenin with nuclear T cell factor (TCF) transcription factors. We identified target genes of the Wnt/beta-catenin/TCF pathway in the most immature (CD4-CD8-CD34+) thymocytes using Affymetrix DNA microarrays in combination with three different functional assays for in vitro induction of Wnt signaling. A relatively small number (approximately 30) of genes changed expression, including several proliferation-inducing transcription factors such as c-fos and c-jun, protein phosphatases, and adhesion molecules, but no genes involved in differentiation to mature T cell stages. The adhesion molecules likely confine the proliferating immature thymocytes to the appropriate anatomical sites in the thymus. For several of these target genes, we validated that they are true Wnt/beta-catenin/TCF target genes using real-time quantitative PCR and reporter gene assays. The same core set of genes was repressed in Tcf-1-null mice, explaining the block in early thymocyte development in these mice. In conclusion, Wnt signals mediate proliferation and cell adhesion, but not differentiation of the immature thymic progenitor pool.

Animals↗
PubMed1991

Low level of Hox1.3 gene expression does not preclude the use of promoterless vectors to generate a targeted gene disruption. off.

A variety of experimental approaches have been devised recently to mutate mammalian genes by homologous recombination. In this report, we describe the disruption of the Hox1.3 locus by using two of these approaches, namely, positive-negative selection and activation of a promoterless gene. Interestingly, we observe similarly high frequencies of targeted disruption with both procedures. The high frequency of targeted disruption with a promoterless vector was unexpected given the extremely low level of Hox1.3 expression in the embryonic stem cell line used for these studies. These data indicate that minimal expression of the target gene is required to enrich for homologous recombination events with promoterless vectors and thus enhance the promoterless gene approach as a general strategy to mutate mammalian genes by homologous recombination.

Animals↗
PubMed2006

Gfi1b alters histone methylation at target gene promoters and sites of gamma-satellite containing heterochromatin.

Gfi1b is a 37 kDa nuclear protein with six C2H2 zinc-finger domains that can silence transcription upon binding to specific target gene promoters. Here we show by using a chromatin immunoprecipitation and cloning protocol that Gfi1b also binds to gamma-satellite sequences that mainly occur in pericentric heterochromatin. Immuno-FISH experiments demonstrated that Gfi1b is localized at foci of pericentric heterochromatin identified by DAPI staining. Elevated levels of Gfi1b correlated with increased histone H3 lysine 9 dimethylation at sites neighboring gamma-satellite sequences but also at Gfi1b target gene promoters. In Gfi1b-deficient cells, however, a decrease of histone H3 lysine 9 trimethylation and a loss of heterochromatic structures was observed. Strikingly, we found that Gfi1b binds to both SUV39H1 and G9A histone methyl transferases, which provides a direct link between histone methylation and Gfi1b at heterochromatic and euchromatic sites. We propose that Gfi1b functions in heterochromatin formation and silencing of euchromatic transcription by recruiting histone methyl transferases to either gamma-satellite sequences or specific target gene promoters.

Animals↗
PubMed2006

Tumor-specific apoptotic gene targeting overcomes radiation resistance in esophageal adenocarcinoma.

PURPOSE: To overcome radiation resistance in esophageal adenocarcinoma by tumor-specific apoptotic gene targeting using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). METHODS AND MATERIALS: Adenoviral vector Ad/TRAIL-F/RGD with a tumor-specific human telomerase reverse transcription promoter was used to transfer TRAIL gene to human esophageal adenocarcinoma and normal human lung fibroblastic cells (NHLF). Activation of apoptosis was analyzed by Western blot, fluorescent activated cell sorting, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate labeling (TUNEL) assay. A human esophageal adenocarcinoma mouse model was treated with intratumoral injections of Ad/TRAIL-F/RGD plus local radiotherapy. RESULTS: The combination of Ad/TRAIL-F/RGD and radiotherapy increased the cell-killing effect in all esophageal adenocarcinoma cell lines but not in NHLF cells. This combination also significantly reduced clonogenic formation (p < 0.05) and increased sub-G1 deoxyribonucleic acid accumulation in cancer cells (p < 0.05). Activation of apoptosis by Ad/TRAIL-F/RGD plus radiotherapy was demonstrated by activation of caspase-9, caspase-8, and caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase in vitro and TUNEL assay in vivo. Combined Ad/TRAIL-F/RGD and radiotherapy dramatically inhibited tumor growth and prolonged mean survival in the esophageal adenocarcinoma model to 31.6 days from 16.7 days for radiotherapy alone and 21.5 days for Ad/TRAIL-F/RGD alone (p < 0.05). CONCLUSIONS: The combination of tumor-specific TRAIL gene targeting and radiotherapy enhances the effect of suppressing esophageal adenocarcinoma growth and prolonging survival.

Adenocarcinoma↗
PubMed2004

The common and distinct target genes of the p53 family transcription factors.

p53 is the most commonly mutated gene in human cancer. After activation by cellular stresses such as DNA damage or oncogene activation, p53, a sequence-specific DNA-binding protein, induces the expression of target genes which mediate tumor suppression. Two recently identified p53 homologues, p63 and p73, appear to function similarly to p53, that is, they both activate target gene expression and suppress cell growth when overexpressed; however, the p63 and p73 genes are rarely mutated in human cancer and do not adhere to Knudson's classical model of a tumor suppressor gene. Recently, exciting observations suggest nonoverlapping functions for the family members. Herein, we outline the recent literatures identifying and characterizing both the common and distinct target genes of the p53 family transcription factors in relation to their signaling pathways.

Apoptosis↗
PubMed2001

Multi-functional roles of Stat3 revealed by conditional gene targeting.

Signal transducer and activator of transcription (STAT) is a family of transcription factors composed of seven members. Gene-targeted mice of each STAT family protein displayed defective responses to cytokines, demonstrating an important role in cytokine-mediated biological responses. However, unlike the mice lacking other STAT proteins, Stat3-deficient mice died during their early embryogenesis. Therefore, in an attempt to avoid the lethality and assess the role of Stat3 in cytokine-mediated functions in mouse adult tissues, conditional gene targeting utilizing a Cre-loxP system was achieved. By this method, Stat3 was disrupted in several types of tissue, including T cells, macrophages, skin, and mammary gland. Analyses of these Stat3-mutant mice revealed important roles of Stat3 in biological functions in each tissue.

Animals↗
PubMed2005

The transcriptional coactivator Yes-associated protein drives p73 gene-target specificity in response to DNA Damage.

The transcriptional coactivator Yes-associated protein (YAP) has been shown to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show that YAP requires the promyelocytic leukemia gene (PML) and nuclear body localization to coactivate p73. YAP imparts selectivity to p73 by promoting the activation of a subset of p53 and/or p73 target promoters. Endogenous p73, YAP, and p300 proteins are concomitantly recruited onto the regulatory regions of the apoptotic target gene p53AIP1 only when cells are exposed to apoptotic conditions. Silencing of YAP by specific siRNA impairs p300 recruitment and reduces histone acetylation on the p53AIP1 target gene, resulting in delayed or reduced apoptosis mediated by p73. We also found that YAP contributes to the DNA damage-induced accumulation of p73 and potentiates the p300-mediated acetylation of p73. Altogether, our findings identify YAP as a key determinant of p73 gene targeting in response to DNA damage.

Acetylation↗