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Results for “targeted integration”

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PubMed2005

piggyBac internal sequences are necessary for efficient transformation of target genomes.

A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full-length or less extensive internal deletion constructs. We have re-examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3-ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5' and 3' terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL-BacII-ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3' internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.

Animals↗

PubMed2000

The Rad51 paralog Rad51B promotes homologous recombinational repair.

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B(-/-) cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B(-/-) cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to gamma-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B(-/-) cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.

Amino Acid Sequence↗

PubMed2006

Behavioral reference frames for planning human reaching movements.

At some stage in the process of a sensorimotor transformation for a reaching movement, information about the current position of the hand and information about the location of the target must be encoded in the same frame of reference to compute the hand-to-target difference vector. Two main hypotheses have been proposed regarding this reference frame: an eye-centered and a body-centered frame. Here we evaluated these hypotheses using the pointing errors that subjects made when planning and executing arm movements to memorized targets starting from various initial hand positions while keeping gaze fixed in various directions. One group of subjects (n = 10) was tested without visual information about hand position during movement planning (unseen-hand condition); another group (n = 8) was tested with hand and target position simultaneously visible before movement onset (seen-hand condition). We found that both initial hand position and gaze fixation direction had a significant effect on the magnitude and direction of the pointing error. Errors were significantly smaller in the seen-hand condition. For both conditions, though, a reference frame analysis showed that the errors arose at an eye- or hand-centered stage or both, but not at a body-centered stage. As a common reference frame is required to specify a movement vector, these results suggest that an eye-centered mechanism is involved in integrating target and hand position in programming reaching movements. We discuss how simple gain elements modulating the eye-centered target and hand-position signals can account for these results.

Adult↗

PubMed1998

Homologous recombination, but not DNA repair, is reduced in vertebrate cells deficient in RAD52.

Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52(-/-) mutants of the chicken B-cell line DT40. Surprisingly, RAD52(-/-) cells were not hypersensitive to DNA damages induced by gamma-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52(-/-) cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52(-/-) cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.

Animals↗

PubMed1996

Isolation and characterization of human hepatoma cells with targeted insertions of a gpt selectable marker in the alpha 1-antitrypsin locus.

The bacterial xanthine-guanine phosphoribosyl transferase (gpt) gene was inserted by homologous recombination into the chromosomal alpha 1-antitrypsin (alpha 1AT) gene of HPRT-deficient human hepatoma cells. These insertions encoded chimeric alpha 1AT-gpt mRNAs that were expressed in the modified cells. Six targeted integrations were obtained, but only two of these harbored simple insertion events. The remaining four homologous insertions contained additional DNA sequences 3' of the gpt coding cassette. Variant cell lines deficient for gpt expression were isolated from transfectants containing either homologous or non-homologous gpt insertions by selection in media containing 6-thioguanine. These variant cell lines expressed alpha 1AT but not alpha 1AT-gpt mRNAs, indicating that they contained expression defects in cis. Genotypic analyses suggested that the predominant mechanism by which the variants were generated was by nondisjunctive loss of chromosomes containing the modified alpha 1AT-gpt alleles. Somatic cell hybrids formed by fusing hepatoma cells containing targeted alpha 1AT-gpt insertions with fibroblasts exhibited extinction of both modified and unmodified alpha 1AT alleles.

Base Sequence↗

PubMed2002

Novel ATPase of SNF2-like protein family interacts with androgen receptor and modulates androgen-dependent transcription.

Nuclear receptors, including the androgen receptor (AR), regulate target cell transcription through interaction with auxiliary proteins to modify chromatin structure. We describe herein a novel AR-interacting protein, termed ARIP4, that has structural features typical of the SNF2-like protein family. With regard to the Snf2 domain, the closest homolog of ARIP4 is the ATRX protein. ARIP4 is a nuclear protein and comprises 1466 amino acids. It interacts with AR in vitro and in cultured yeast and mammalian cells. ARIP4 can be labeled with 8-azido-[gamma-32P]ATP and exhibits DNA-dependent ATPase activity. Like several ATP-dependent chromatin remodeling proteins, ARIP4 generates superhelical torsion within linear DNA fragments in an ATP-dependent manner. With a stably integrated target promoter, ARIP4 elicits a modest enhancement of AR-dependent transactivation. In transient cotransfection assays, ARIP4 modulates AR function in a promoter-dependent manner; it enhances receptor activity on minimal promoters, but does not activate more complex promoters. ARIP4 mutants devoid of ATPase activity fail to alter DNA topology and behave as trans-dominant negative regulators of AR function in transient assays.

Adenosine Triphosphatases↗

PubMed2002

Arg tyrosine kinase is involved in homologous recombinational DNA repair.

c-Abl plays important roles in cellular response to DNA damage. However, possible roles for Arg (Abl-related gene) in DNA damage response are unknown. Here, we show that ionizing radiation (IR)-induced Rad51 focus formation is reduced in Arg-deficient cells generated from a chicken B cell line by targeted disruption. This is consistent with the findings that Arg-deficient cells display hypersensitivity to IR, elevated frequencies of IR-induced chromosomal aberrations, and reduced targeted integration frequencies. All of these abnormalities in DNA damage repair are also observed in ATM-deficient cells but not in c-Abl-deficient cells. Finally, we show that Arg interacts with and phosphorylates Rad51 in 293T cells. These results suggest that Arg plays a role in homologous recombinational (HR) DNA repair by phosphorylating Rad51.

Animals↗

PubMed2025

ImmunoTar-integrative prioritization of cell surface targets for cancer immunotherapy.

MOTIVATION: Cancer remains a leading cause of mortality globally. Recent improvements in survival have been facilitated by the development of targeted and less toxic immunotherapies, such as chimeric antigen receptor (CAR)-T cells and antibody-drug conjugates (ADCs). These therapies, effective in treating both pediatric and adult patients with solid and hematological malignancies, rely on the identification of cancer-specific surface protein targets. While technologies like RNA sequencing and proteomics exist to survey these targets, identifying optimal targets for immunotherapies remains a challenge in the field. RESULTS: To address this challenge, we developed ImmunoTar, a novel computational tool designed to systematically prioritize candidate immunotherapeutic targets. ImmunoTar integrates user-provided RNA-sequencing or proteomics data with quantitative features from multiple public databases, selected based on predefined criteria, to generate a score representing the gene's suitability as an immunotherapeutic target. We validated ImmunoTar using three distinct cancer datasets, demonstrating its effectiveness in identifying both known and novel targets across various cancer phenotypes. By compiling diverse data into a unified platform, ImmunoTar enables comprehensive evaluation of surface proteins, streamlining target identification and empowering researchers to efficiently allocate resources, thereby accelerating the development of effective cancer immunotherapies. AVAILABILITY AND IMPLEMENTATION: Code and data to run and test ImmunoTar are available at https://github.com/sacanlab/immunotar.

Humans↗

PubMed1995

Co-existence of NADPH-diaphorase, fibroblast growth factor-2 and fibroblast growth factor receptor in spinal autonomic system suggests target-specific actions.

In the rat spinal cord, we found substantial co-existence of fibroblast growth factor-2, fibroblast growth factor receptor (type-1 or flg) immunoreactivity and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity (a histochemical marker for neuronal nitric oxide synthase) in preganglionic autonomic cell groups of intermediate layers VI, VII and X. Anti-fibroblast growth factor-2 and anti-nitric oxide synthase binding sites were confined to the cytoplasm of reactive neurons as judged by immunogold electron microscopy. Within the major autonomic nucleus, i.e. intermediolateral column, three different populations were identified: (i) fibroblast growth factor and fibroblast growth factor receptor, (ii) fibroblast growth factor/NADPH-diaphorase and (iii) NADPH-diaphorase-only stained cell groups. Sympathoadrenal neurons were prelabelled with fluorescent tracer Fast Blue and co-stained for fibroblast growth factor-like protein and NADPH-diaphorase, suggesting heterologous diversification of neuronal phenotypes and functional organization in the spinal autonomic system. Our findings suggest intriguing roles for nitric oxide and fibroblast growth factor-2 cytokine in the preganglionic sympathetic spinal cord system: The "short-term" diffusible messenger nitric oxide may act as "tonic" and/or "phasic" signal within rostrocaudally oriented function-specific preganglionic units necessary for integrated target control. The "long-term" messenger fibroblast growth factor-2 may be involved in, for example, cytokine-dependent regulation of neuronal NADPH-diaphorase/nitric oxide synthase. Furthermore, co-existence of NADPH-diaphorase, fibroblast growth factor-2 and receptor in sympathoadrenal neurons suggest mutual target-specific regulatory functions, e.g. hormone release and blood perfusion or maintenance of phenotype and plasticity responsiveness of adrenal medullary tissue.

Animals↗

PubMed1982

Methods of exercise testing and training prescription for cardiovascular diseases.

Physical exercise testing and training in health and in heart disease may differ in concepts, targets and interpretation. Untrained males (n = 1962) of different age (18-65 years)--638 apparently healthy, 121 with arterial hypertension stage I-II, 1140 after confirmed acute myocardial infarction, and 63 after mitral surgery--were submitted to near-maximal bicycle spiro-ergometry (sitting, 60 rpm). Correlations between oxygen uptake (VO2), external work (W), heart rate (HR), systolic blood pressure (BP), double product (HR x BD = DP, as an indirect index of myocardial oxygen uptake), and other derived indices, varied substantially. The mutual predictive power of non-invasive cardiocirculatory parameters was weak particularly at low and moderate exercise (50-100 Watts). Different factors of priority influenced the variety in regression equations obtained in the separate groups. Discriminant values and limits rather than "norms" seem more appropriate in order to select the most relevant intervention (physical training, medical treatment, surgery). "Optimization" of disturbed inter-factor relations (e.g. of HR, BP, DP, W, VO2, VO2/kg, mechanical efficiency) may define the integral target of physical training in cardiac patients, if indicated. "Target-oriented" exercise prescription is discussed with examples. The cardiac cost of physical work (DP/VO2) is assessed in various heart conditions with its implications on exercise prescription. An attempt is made at differentiating between training the patient and "training" the diseased heart, and some controversies in terminology and in practical approach are stressed.

Adult↗

PubMed1998

A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris.

The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial beta-glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells. GenBank Accession Numbers are: pIB1, AF027958; pIB2, AF0279959; pIB3, AF027960; pIB4, AF027961.

Alcohol Oxidoreductases↗

PubMed2001

Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs.

The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.

Animals↗

PubMed1985

The auditory corticopontocerebellar projection in the rat: inputs to the paraflocculus and midvermis. An anatomical and physiological study.

This study investigated afferent projections to the cerebellum, in particular those from the auditory cerebral cortex. The parafloccular lobule of the rat cerebellum is shown to be a primary target for the auditory cortical information with the midvermal region being a receiving area from the inferior colliculus. The method of anterograde transport of tritiated amino acids was employed to determine projections of the auditory cortex to the pons. Autoradiography showed that the site of termination of efferents from the auditory cortex corresponds to the location of neurons that project to the paraflocculus. Histogram analysis of neuronal activity in halothane anesthetized rats was employed to determine the response characteristics of neurons in paraflocculus and midvermis following cortical and tectal electrical stimulation. In addition, unit recordings of parafloccular neurons in immobilized, locally anesthetized animals demonstrated general characteristics of the responses of these neurons to auditory field stimulation. Electrical stimulation of the auditory cortex evoked mixed, excitatory-inhibitory and pure inhibitory mossy fiber responses in 33% of neurons in the paraflocculus, with no responses evident in the midvermis. Following inferior collicular stimulation, 12.6% of the neurons in the midvermis elicited a response. Recordings from parafloccular neurons in unanesthetized, immobilized rats showed evidence for excitatory and inhibitory mossy fiber responses, following auditory field stimulation. This spectrum of basic studies establishes the existence of a pathway in which the paraflocculus is revealed as an integrating target for cortical auditory information.

Animals↗

PubMed1995

Targeted disruption of an H3-IV/H3-V gene pair causes increased expression of the remaining H3 genes in the chicken DT40 cell line.

The chicken H3 gene family contains 12 members (H3-I to H3-XII). Nine of them belong to two major histone gene clusters, but the genomic locations of the others are unknown. In the DT40 chicken B cell line, H3-IV and H3-V, which are located in inverted orientation and share a 3'-untranslated region of 531 bp, produce about 24% of the steady-state level of total mRNAs from all the H3 genes. To study the nature of these two genes, we carried out targeted integration into DT40 cells of constructs of the H3-IV/H3-V locus. Analysis of the stable transfectants showed that the growth rate of DT40 cells was unchanged in the absence of two copies of the H3-IV/H3-V locus. Furthermore, the remaining H3 genes were shown to be expressed more in the mutants lacking one or two H3-IV/H3-V alleles than DT40 cells, and the steady-state level of total H3 mRNAs remained constant in all the mutant cell lines tested. The levels of mRNAs from the H2B and H1 genes, which are located close to the H3 genes, were unchanged. These results demonstrate that the chicken H3 gene family has the ability to compensate for disruption of H3-IV/H3-V, which are normally responsible for about 24% of total H3 mRNAs.

Alleles↗

PubMed1999

Gene targeting in Penicillium chrysogenum: disruption of the lys2 gene leads to penicillin overproduction.

Two strategies have been used for targeted integration at the lys2 locus of Penicillium chrysogenum. In the first strategy the disruption of lys2 was obtained by a single crossing over between the endogenous lys2 and a fragment of the same gene located in an integrative plasmid. lys2-disrupted mutants were obtained with 1.6% efficiency when the lys2 homologous region was 4.9 kb, but no homologous integration was observed with constructions containing a shorter homologous region. Similarly, lys2-disrupted mutants were obtained by a double crossing over (gene replacement) with an efficiency of 0.14% by using two lys2 homologous regions of 4.3 and 3.0 kb flanking the pyrG marker. No homologous recombination was observed when the selectable marker was flanked by short lys2 homologous DNA fragments. The disruption of lys2 was confirmed by Southern blot analysis of three different lysine auxotrophs obtained by a single crossing over or gene replacement. The lys2-disrupted mutants lacked alpha-aminoadipate reductase activity (encoded by lys2) and showed specific penicillin yields double those of the parental nondisrupted strain, Wis 54-1255. The alpha-aminoadipic acid precursor is channelled to penicillin biosynthesis by blocking the lysine biosynthesis branch at the alpha-aminoadipate reductase level.

Aldehyde Oxidoreductases↗

PubMed2000

Combinatorial chemistry and contemporary pharmacology.

Both solid- and liquid-phase combinatorial chemistry have emerged as powerful tools for identifying pharmacologically active compounds and optimizing the biological activity of a lead compound. Complementary high-throughput in vitro assays are essential for compound evaluation. Cell-based assays that use optical endpoints permit investigation of a wide variety of functional properties of these compounds including specific intracellular biochemical pathways, protein-protein interactions, and the subcellular localization of targets. Integration of combinatorial chemistry with contemporary pharmacology now represents an important factor in drug discovery and development.

Combinatorial Chemistry Techniques↗

PubMed1992

Response of retinal terminals to loss of postsynaptic target neurons in the dorsal lateral geniculate nucleus of the adult cat.

We have used the neurotoxin kainic acid to produce rapid degeneration of neurons in the dorsal lateral geniculate nucleus (dLGN) of the adult cat. This degeneration mimics the rapid loss of geniculate neurons seen after visual cortex ablation in the neonate. Subsequent anterograde transport of horseradish peroxidase injected into the eye was used to reveal the projection patterns of retinal ganglion cell axons at different survival periods after the kainic acid injection. The density of retinal projections to the degenerated regions of the geniculate was reduced considerably at 4 and 6 months survival, but at 2 months was not significantly different from normal. The laminar pattern of projections to degenerated regions of the geniculate did not change in any animals studied, even when an adjacent lamina contained surviving cells. Electron microscopic examination of degenerated dLGN revealed intact retinal (RLP) and RSD terminals at all survival times, although the density of terminals appeared much reduced when compared to controls. Some RLP terminals exhibited the "dark reaction" of degeneration and these degenerating terminals were most numerous at 2 months survival. These findings demonstrate that, in response to degeneration of their usual target cells, mature retinal ganglion cells with withdraw their axon terminals from these regions of degeneration. We conclude that mature retinal ganglion cells continue to be dependent on target integrity for the maintenance of a normal axonal arborization.

Animals↗

PubMed2004

Immunoglobulin gene conversion: insights from bursal B cells and the DT40 cell line.

Chicken B cells diversify their immunoglobulin (Ig) light and heavy chain genes by pseudogene templated gene conversion within the bursa of Fabricius. Although Ig gene conversion was initially believed to occur only in birds, it is now clear that most farm animals also use this elegant mechanism to develop an immunoglobulin gene repertoire. The best model to study Ig gene conversion remains the chicken Ig light chain locus due to its compact size and the fact that all the pseudogene donors are sequenced. Furthermore, gene conversion continues in the bursa-derived DT40 cell line whose genome can be easily modified by targeted integration of transfected constructs. Disruption of the AID gene, which had been shown to control somatic hypermutation and switch recombination in mammals leads to a complete block of gene conversion in DT40 indicating that all B-cell specific repertoire formation is controlled by the same gene. Here, we review the genetics and the molecular mechanism of Ig gene conversion based on sequence analysis of bursal B cells and gene disruption studies in the DT40 cell line.

Animals↗