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1.
Allan Bradley 《Current opinion in biotechnology》1991,2(6):823-829
Over the past year, gene targeting in mammalian cells has become a facile technology. By using a variety of selection and screening protocols, it has become possible to direct modifications at the nucleotide level to specific genes, to target marker genes so that they become expressed under the control of endogenous promoters and to delete large regions of the genome. 相似文献
2.
In order to facilitate the procedure of mammalian gene targeting, we have produced and functionally tested a series of generic vectors. Homologous recombination has been achieved with each vector. The vectors are designed for both replacement and insertional recombination, are suitable for hit and run strategies and contain all necessary genetic elements for both positive-negative and promoterless/gene fusion enrichment of homologous integrations. Multiple unique restriction sites are included to simplify the incorporation of genomic targeting sequences. 相似文献
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4.
The mammalian DP, RB-like, E2F, and MuvB-like proteins (DREAM) complex, whose key components include p130 and E2F4, plays a fundamental role in repression of cell cycle-specific genes during growth arrest. Mammalian DREAM is well conserved with Drosophila and Caenorhabditis elegans complexes that repress pivotal developmental genes, but the mammalian complex has been thought to exist only in quiescent cells and not to be linked with development. However, new findings here identify tissue-specific promoters repressed by DREAM in proliferating precursors, revealing a new connection between control of growth arrest and terminal differentiation. Mechanistically, tissue-specific promoter occupation by DREAM is dependent on the integrity of a repressor form of the SWI/SNF chromatin-remodeling complex. 相似文献
5.
Kitagawa Y Tani H Limn CK Matsunaga TM Moriishi K Matsuura Y 《Journal of virology》2005,79(6):3639-3652
The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) can infect a variety of mammalian cells, as well as insect cells, facilitating its use as a viral vector for gene delivery into mammalian cells. Glycoprotein gp64, a major component of the budded AcMNPV envelope, is involved in viral entry into cells by receptor-mediated endocytosis and subsequent membrane fusion. We examined the potential production of pseudotype baculovirus particles transiently carrying ligands of interest in place of gp64 as a method of ligand-directed gene delivery into target cells. During amplification of a gp64-null pseudotype baculovirus carrying a green fluorescent protein gene in gp64-expressing insect cells, however, we observed the high-frequency appearance of a replication-competent virus incorporating the gp64 gene into the viral genome. To avoid generation of replication-competent revertants, we prepared pseudotype baculoviruses by transfection with recombinant bacmids without further amplification in the gp64-expressing cells. We constructed gp64-null recombinant bacmids carrying cDNAs encoding either vesicular stomatitis virus G protein (VSVG) or measles virus receptors (CD46 or SLAM). The VSVG pseudotype baculovirus efficiently transduced a reporter gene into a variety of mammalian cell lines, while CD46 and SLAM pseudotype baculoviruses allowed ligand-receptor-directed reporter gene transduction into target cells expressing measles virus envelope glycoproteins. Gene transduction mediated by the pseudotype baculoviruses could be inhibited by pretreatment with specific antibodies. These results indicate the possible application of pseudotype baculoviruses in ligand-directed gene delivery into target cells. 相似文献
6.
In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism. 相似文献
7.
《Genomics》2020,112(1):304-311
Genetic changes in Hypoxanthine guanine phosphoribosyltransferace (HPRT1) gene can alter the expression of the dopamine neurotransmitter leads to abnormal neuron function, a disease called Lesch-Nyhan syndrome (LNS). Although different studies were conducted on LNS, information on codon usage bias (CUB) of HPRT1 gene is limited. The present study examines the genetic determinants of CUB in HPRT1 gene using twelve mammalian species. In the coding sequence of HPRT1 genes, A/T ending codons was most frequently used. A higher ENC value was observed indicating lower HPRT1 gene expression in the selected mammalian species. Correlation analysis indicates that compositional constraints under mutation pressure can involve in CUB of HPRT1 genes among the selected mammalian species. Relative synonymous codon usage (RSCU) value revealed that the codons such as ACT, AGG, ATT and AGC were over-represented in each of the mammalian species. Result from the analysis of the RSCU indicates that compositional constraint is a key driver for the variation in codon usage. Ratio of nonsynonymous (dN) and synonymous (dS) substitution further suggested that purifying selection occurs among the HPRT1 gene of studied mammals to maintain its protein function under the process of evolution. Our findings report an insight into the codon usage patterns of HPRT1 gene and will be useful for LNS management. 相似文献
8.
To better understand the mechanism of homologous recombination in mammalian cells that facilitates gene targeting, we have analyzed the recombination reaction that inserts a plasmid into a homologous chromosomal locus in mouse embryonic stem cells. A partially deleted HPRT gene was targeted with various plasmids capable of correcting the mutation at this locus, and HPRT+ recombinants were directly selected in HAT medium. The structures of the recombinant loci were then determined by genomic Southern blot hybridizations. We demonstrate that plasmid gaps of 200, 600, and 2,500 bp are efficiently repaired during the integrative recombination reaction. Targeting plasmids that carry a double-strand break or gap in the region of DNA homologous to the target locus produce 33- to 140-fold more hypoxanthine-aminopterin-thymidine-resistant recombinants than did these same plasmids introduced in their uncut (supercoiled) forms. Our data suggest that double-strand gaps and breaks may be enlarged prior to the repair reaction since sequence heterologies carried by the incoming plasmids located close to them are often lost. These results extend the known similarities between mammalian and yeast recombination mechanisms and suggest several features of the insertional (O-type) gene targeting reaction that should be considered when one is designing mammalian gene targeting experiments. 相似文献
9.
Nielsen ML de Jongh WA Meijer SL Nielsen J Mortensen UH 《Applied and environmental microbiology》2007,73(22):7240-7245
Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of pyrG. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed, pyrG function can be rapidly returned to wild type by a simple selection scheme. 相似文献
10.
Pea (Pisum sativum L.) satellited chromosome reconstructions were analyzed by cytologic markers to identify segregation distortion events. The presence of modified chromosomes was evaluated on the basis of additional rDNA genes, an extra and a longer satellite, all derived from chromosome 5 and chromosome 7 from P. fulvum Sibth. & Sm. The segregation of modified satellited chromosome 5 was monitored through fluorescent in situ hybridization with rDNA probe; it fitted the expected 1:2:1 ratio after self-pollination of a heterozygous genotype for modified chromosome 5. In different genotypes, which were heterozygous for both modified chromosomes 5 and 7, the combined segregation of these chromosomes showed the occurrence of seven karyotype classes instead of the expected nine. The classes with modified chromosome 7 and without modified chromosome 5, whether heterozygous or homozygous, were absent. The hypothesis of gamete selection was rejected since the expected segregation ratio of 5:3:1 was significant by chi-square test. Based on the other hypothesis of postzygotic selection, the segregation ratio did not show a significant deviation from the expected 9:3:1 ratio, thereby indicating that embryo abortion caused the segregation distortion (SD). The hypothesis of the SD system involving two loci carried by the alien satellites of modified chromosomes 5 and 7 is discussed in relation to the evolution of the P. fulvum genome. 相似文献
11.
Nielsen ML Albertsen L Lettier G Nielsen JB Mortensen UH 《Fungal genetics and biology : FG & B》2006,43(1):54-64
The rapid accumulation of genomic sequences from a large number of eukaryotes, including numerous filamentous fungi, has created a tremendous scientific potential, which can only be realized if precise site-directed genome modifications, like gene deletions, promoter replacements, in-frame GFP fusions and specific point mutations can be made rapidly and reliably. The development of gene-targeting techniques in filamentous fungi and other higher eukaryotes has been hampered because foreign DNA is predominantly integrated randomly into the genome. For Aspergillus nidulans, we have developed a flexible method for gene-targeting employing a bipartite gene-targeting substrate. This substrate is made solely by PCR, which obviates the need for bacterial subcloning steps. The method reduces the number of false positives and can be used to produce virtually any genome alteration. A major advance of the method is that it allows multiple subsequent genome manipulations to be performed as the selectable marker is recycled. 相似文献
12.
X-ray diffraction patterns from mammalian heart muscle 总被引:8,自引:0,他引:8
We have obtained light and X-ray diffraction patterns from trabecular and papillary muscles of various mammalian hearts in the living resting state and in rigor. Equatorial X-ray diffraction patterns from living muscles show the 1,0 and 1,1 reflections from a hexagonal lattice of filaments. The lattice spacing varies with sarcomere length over the observable range (2·0 to 2·5 μm) in such a manner that the lattice volume remains constant. In the living resting state the 1,0 reflection is stronger than the 1,1 reflection, whereas in rigor the 1,1 reflection is almost as strong as the 1,0 reflection. These intensity changes are similar to those found in vertebrate skeletal muscle, suggesting that the mechanism of cross-bridge attachment to actin is similar in both muscles.Two types of meridional X-ray diffraction pattern were observed in muscles in different conditions. One type, obtained from dead or glycerol-extracted muscles or from muscles treated with iodoacetate, showed a strong actin-related pattern but only a weak pattern associated with myosin. This type of pattern was similar to that from vertebrate skeletal muscle in rigor. The other type, obtained from living, resting muscle, showed a weaker actin pattern but a stronger myosin pattern. The myosin pattern included layer-line reflections associated with projections from the thick filaments. This second type of pattern was similar to that from resting vertebrate skeletal muscle, but the layer lines were weaker. The weakness of the myosin layer lines may indicate that part of the high resting tension found in heart muscle arises from a small amount of actin-myosin interaction in the resting state. Such interaction could provide a mechanism for varying the diastolic length of heart muscle and thereby the diastolic volume of the heart. 相似文献
13.
G Del Boccio E Casalone P Sacchetta A Pennelli C Di Ilio 《Biochemical medicine and metabolic biology》1986,36(3):306-312
The occurrence of glutathione transferase isoenzymes in mammalian erythrocytes was investigated. The enzymes present in the hemolysates of human, horse, beef, pig, and sheep erythrocytes were purified by a column of GSH-linked epoxy-activated Sepharose 6B and subjected to an isoelectric focusing run in the pH range 3.5-10. Human and horse preparations were resolved in a single peak of activity centered at pH 4.6 and 5.9, respectively. Two forms with a maximum of activity at pH 4.9 and 7.0 and four with a maximum at pH 5.9, 6.5, 7.1, and 8.1 were separated from bovine and porcine erythrocytes. At least six forms ranging from pH 4.3 to pH 7.1 were present in the ovine preparation, the neutral contributing more than 90% of total activity. The subunit composition of affinity-bound fractions was studied by sodium dodecyl sulfate-gel electrophoresis. The analysis revealed that erythrocyte glutathione transferases are composed of subunits of identical molecular weights. This result suggests that the polymorphism existing in beef, pig, and sheep may be due to charge isomers. The erythrocyte glutathione transferases did not express selenium-independent GSH peroxidase activity. 相似文献
14.
Sergei Vadimovich Evsikov Ludmila Michailovna Morozova Alexander Petrovich Solomko 《Development genes and evolution》1994,203(4):199-204
A new micromanipulation technique permitted the scrambling of the zygote cytoplasm. Such interference had no effect on preimplantation development, and when zygotes with scrambled cytoplasm were transfered to the pseudopregnant females, normal and fertile mice were born. This demonstrates that no morphogenetic factors are prelocalized in the egg cytoplasm. Cleavage characteristics of mouse embryos provide the evidence that zygote cytoplasm does not define any determinate type of cleavage. We conclude that the mechanism of ooplasmic segregation is not used in the mouse (and presumably mammalian) development. It is suggested that the turning point in the evolution of mammalian embryogenesis was the transition to the intrauterine development, that started the process leading among other changes, to the loss of the ooplasmic morphogenetic determinants.
Correspondence to: S.V Evsikov 相似文献
15.
Self-fertile apple resulting from S-RNase gene silencing 总被引:3,自引:0,他引:3
Self-incompatibility (SI) restricts fertilisation and fruit setting in many tree fruit crops. In apple, we have produced transgenic trees harbouring extra copies of the endogenous S-gene controlling SI. Two independent transgenic genotypes were characterised in detail. Controlled self- and cross-pollination of the flowers of trees from both genotypes over a 3-year-period showed that the transgenic lines produced normal levels of fruit and seeds after selfing. In contrast, the controls produced much less fruit following self- compared to cross-pollination. Fruit set data correlated with the results of microscopic evaluation of pollen tube growth through the pistil, which revealed inhibition after selfing in the controls but not in the transgenic lines. The self-fertile phenotype was associated with the complete absence of pistil S-RNase proteins, which are the products of the targeted S-gene. These results confirm that self-fertility was due to inhibition of expression of the S-RNase gene in the pistil, resulting in un-arrested self-pollen tube growth, and fertilisation.Communicated by P. Debergh 相似文献
16.
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. 相似文献
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18.
DNA interstrand cross-links (ICLs) are critical cytotoxic lesions produced by cancer chemotherapeutic agents such as the nitrogen mustards and platinum drugs; however, the exact mechanism of ICL-induced cell death is unclear. Here, we show a novel mechanism of p53-independent apoptotic cell death involving prolonged cell-cycle (G2) arrest, ICL repair involving HR, transient mitosis, incomplete cytokinesis, and gross chromosomal abnormalities resulting from ICLs in mammalian cells. This characteristic ‘giant'' cell death, observed by using time-lapse video microscopy, was reduced in ICL repair ERCC1- and XRCC3-deficient cells. Collectively, the results illustrate the coordination of ICL-induced cellular responses, including cell-cycle arrest, DNA damage repair, and cell death. 相似文献
19.
Rafael P. Vázquez-Manrique James C. Legg Birgitta Olofsson Sung Ly Howard A. Baylis 《Genomics》2010,95(1):37-46
Gene targeting is widely used for the precise manipulation of genes. However, in the model organism Caenorhabditis elegans non-transposon mediated gene targeting remains laborious, and as a result has not been widely used. One obstacle to the wider use of this approach is the difficulty of identifying homologous recombination events amongst non-specific events. To improve gene targeting in C. elegans, we used a counter-selection approach to reduce the number of false positives; this involved using unc-119 as a positive-selection marker and GFP as a counter-selection marker which is lost during homologous recombination. This method of gene targeting allows straightforward screening for homologous events using a dissecting microscope equipped for fluorescence. In addition, to improve the final engineered product, we utilised Flp recombinase to remove the unc-119 selection marker, in somatic cells, producing clean knockouts in these cells. Using this strategy we have produced a knockout of the plc-4 gene, which encodes phospholipase C-δ in C. elegans, and demonstrated that conditional gene knockout is feasible in C. elegans. 相似文献
20.
Targeted gene repair, a form of oligonucleotide-directed mutagenesis, employs end-modified single-stranded DNA oligonucleotides to mediate single-base changes in chromosomal DNA. In this work, we use a specific 72-mer to direct the repair of a mutated eGFP gene stably integrated in the genome of DLD-1 cells. Corrected cells express eGFP that can be identified and quantitated by FACS. The repair of this mutant gene is dependent on the presence of a specifically designed oligonucleotide and the frequency with which the mutation is reversed is affected by the induction of DNA damage. We used hydroxyurea, VP16 (etoposide), and thymidine to modulate the rate of DNA replication through the stalling of the replication forks or the introduction of lesions. Addition of hydroxyurea or VP16 before the electroporation of the oligonucleotide, results in an accumulation of double-strand breaks (DSB) whose repair is facilitated by either nonhomologous end joining (NHEJ) or homologous recombination (HR). The addition of thymidine results in DNA damage within replication forks, damage that is repaired through the process of homologous recombination. Our data suggest that gene repair activity is elevated when DNA damage induces or activates the homologous recombination pathway. 相似文献