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PubMed2005

Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ mice.

PURPOSE: To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium-specific manner when induced by doxycycline. METHODS: A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (keratin 12 gene) to produce a knock-in Krt12(rtTA/+) mouse line through gene-targeting techniques. The Krt12(rtTA/+) knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12(rtTA/+)/tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow. RESULTS: Administration of doxycycline induced a 15-fold increase of beta-galactosidase activity in the cornea of adult bitransgenic mice (Krt12(rtTA/+)/tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12(rtTA/+) or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of beta-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that beta-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased beta-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet. CONCLUSIONS: A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.

Animals↗

PubMed1991

Comparative study on Rickettsia-like organisms in the midgut epithelial cells of different Glossina species.

The midgut epithelium of Glossina morsitans centralis, G. austeni, G. pallidipes, G. palpalis palpalis, G. p. gambiensis, G. fuscipes fuscipes, G. tachinoides and G. brevipalpis from ILRAD-bred colonies was examined, by electron microscopy, for the presence and distribution of Rickettsia-like organisms (RLOs). RLOs were present in the midgut epithelial cells of all non-teneral tsetse. In G.m. centralis, G. pallidipes and, to a much lesser extent, G. brevipalpis, RLOs were numerous and were present in all the specimens examined. RLOs were present in fewer numbers in the epithelial cells of tenerals of these three tsetse species. In contrast, RLOs occurred in very much lower numbers within the midgut cells of nonteneral G. austeni, G. p. palpalis, G. p. gambiensis, G.f. fuscipes and G. tachinoides; were not seen in every specimen, and were rarely observed in the midgut cells of teneral testse. The RLOs were typical rod-shaped bacteria with an inner and outer membrane, which occurred free within the host cell cytoplasm and appeared to cause no obvious pathology. The micro-organisms divided by binary fission and at least two distinct morphological forms plus a range of intermediate forms were seen in the midgut cells. A comparison of the presence and numbers of RLOs within the midgut cells and the midgut infection rates of both Trypanosoma congolense and T. b. brucei, both between Glossina species and also within the same stock of tsetse, clearly indicates that the ability of trypanosomes to establish and develop to mature infections is unlikely to be correlated solely with the presence of RLOs within the tsetse midgut.

Animals↗

PubMed1998

Evolution of dendritic patterns during alloy solidification: onset of the initial instability.

The evolution of a dendritic pattern from a planar solid-liquid interface during directional solidification of a binary alloy was investigated experimentally. The model alloy used was the transparent organic crystal succinonitrile doped with the laser dye coumarin 152. The buildup of solute ahead of the initially stable planar interface and the subsequent instability of the planar front were measured in detail and compared with recent theoretical calculations by Warren and Langer [Warren, J. A. & Langer, J. S. (1993) Phys. Rev. E 47, 2702- 2712]. The fluorescence of coumarin 152 was used for direct observations of the evolution of the solute concentration profile ahead of the initially planar solid-liquid interface. UV absorption was used to produce thermal perturbations of the sample that generated spatially periodic modulations of the planar interface. This technique allows for measurement of both positive and negative linear growth coefficients (determined from the growth or decay rate of the modulation after the perturbation is switched off) for a large range of wave vectors. Measurements of the evolution of the concentration profile and the linear growth coefficients, and the occurrence of the initial instability, were in good agreement with the Warren-Langer predictions.

Journal Article↗

PubMed2002

In vitro organogenesis and genetic transformation in popular Cucumis sativus L. through Agrobacterium tumefaciens.

The effect of growth regulators and culture conditions on the morphogenetic response of cotyledonary leaf discs was studied in popular cucumber variety (Cucumis sativus cv. Sheetal). Organogenesis was induced directly without any intervening callus phase on Murashige and Skoog medium supplemented with different concentrations of benzyladenine and indole propionic acid. Best results (93%) were obtained in the presence of the 4 mg/L benzyladenine and 1 mg/L IPA. The elongated shoots were rooted in basal medium with 1 mg/L indole butyric acid, hardened and transferred to the field conditions. Genetic transformation system has been established for Cucumis sativus cv. Sheetal, plants by infecting cotyledonary explants with Agrobacterium tumefaciens strain LBA4404 carrying binary plasmid pBI121, which contains scorable marker, beta-glucuronidase and selectable marker nptII under the CaMV 35S promoter. Infection was most effective when explants were infected with Agrobacterium for 15 min and co-cultivated for 2 days in the co-cultivation medium. Shoots were regenerated directly from cotyledonary leaf explants in the presence of kanamycin (50 microg/ml) and analysed. Southern blot analysis confirmed that transformation had occurred. This method will allow genetic improvement of this crop by the introduction of agronomically important genes.

Agrobacterium tumefaciens↗

PubMed2025

Disease candidate genes prediction using positive labeled and unlabeled instances.

Identifying disease genes and understanding their performance is critical in producing drugs for genetic diseases. Nowadays, laboratory approaches are not only used for disease gene identification but also using computational approaches like machine learning are becoming considerable for this purpose. In machine learning methods, researchers can only use two data types (disease genes and unknown genes) to predict disease candidate genes. Notably, there is no source for the negative data set. The proposed method is a two-step process: The first step is the extraction of reliable negative genes from a set of unlabeled genes by one-class learning and a filter based on distance indicators from known disease genes; this step is performed separately for each disease. The second step is the learning of a binary model using causing genes of each disease as a positive learning set and the reliable negative genes extracted from that disease. Each gene in the unlabeled gene's production and ranking step is assigned a normalized score using two filters and a learned model. Consequently, disease genes are predicted and ranked. The proposed method evaluation of various six diseases and Cancer class indicates better results than other studies.

Humans↗

PubMedDate not supplied

Supervised feature ranking using a genetic algorithm optimized artificial neural network.

A genetic algorithm optimized artificial neural network GNW has been designed to rank features for two diversified multivariate data sets. The dimensions of these data sets are 85x24 and 62x25 for 24 or 25 molecular descriptors being computed for 85 matrix metalloproteinase-1 inhibitors or 62 hepatitis C virus NS3 protease inhibitors, respectively. Each molecular descriptor computed is treated as a feature and input into an input layer node of the artificial neural network. To optimize the artificial neural network by the genetic algorithm, each interconnected weight between input and hidden or between hidden and output layer nodes is binary encoded as a 16 bits string in a chromosome, and the chromosome is evolved by crossover and mutation operations. Each input layer node and its associated weights of the trained GNW are systematically omitted once (the self-depleted weights), and the corresponding weight adjustments due to the omission are computed to keep the overall network behavior unchanged. The primary feature ranking index defined as the sum of self-depleted weights and the corresponding weight adjustments computed is found capable of separating good from bad features for some artificial data sets of known feature rankings tested. The final feature indexes used to rank the data sets are computed as a sum of the weighted frequency of each feature being ranked in a particular rank for each data set being partitioned into numerous clusters. The two data sets are also clustered by a standard K-means method and trained by a support vector machine (SVM) for feature ranking using the computed F-scores as feature ranking index. It is found that GNW outperforms the SVM method on three artificial as well as the matrix metalloproteinase-1 inhibitor data sets studied. A clear-cut separation of good from bad features is offered by the GNW but not by the SVM method for a feature pool of known feature ranking.

Algorithms↗

PubMed1994

Perceptual characteristics of binary, trinary, and quaternary odor mixtures consisting of unpleasant constituents.

Among the most obnoxious stimuli that the population at large is exposed to during everyday life are odorous emissions from sewage treatment plants. Such emissions are complex and contain many different types of odorants that vary in quantity depending upon the contents and efficiency of treatment processes. Because little is known about how individual odorants in complex mixtures affect the perception of each other, it is difficult to develop mathematical models that can predict the pleasantness, strength, and quality characteristics of an emission at different distances from a source. In the present study, the interactions of the four major types of odorants emitted by treatment plants worldwide, namely, hydrogen sulphide, isovaleric acid, butanethiol, and skatole, were investigated by measuring the perceived intensity of individual odorants alone and in mixtures, and the overall perceived intensity, unpleasantness, and qualities of mixtures. In addition, models for predicting odor strength were investigated. The results indicated that (i) the perceived odor intensity (odor strength) of mixtures of the odorants was equal or greater than that of any of the individual constituents, but less than the sum of their intensities. However, as the number of components in a mixture increased, the intensity of the most dominant component provided a good approximation of the intensity of the mixture. (ii) The vector model of intensity summation also satisfactorily predicted the odor intensity of mixtures containing two, three, or four of the odorants investigated. (iii) In no instance was the intensity of one odorant enhanced by another, i.e., no synergistic interactions occurred; the greater the number of odorants in a mixture, the greater was the degree of suppression of the individual constituents. (iv) The greater the number of constituents in a mixture the more difficult it became to identify individual constituents. (v) Hydrogen sulphide was the least frequently suppressed constituent, and isovaleric acid and skatole were the most frequently suppressed constituents in mixtures. (vi) The unpleasantness of mixtures was usually greater than that of the individual constituents, indicating that models used for predicting complaint levels in communities affected by sewage odor and based on assumptions related to a single odorant, e.g., hydrogen sulphide, will underestimate the number of complaints. Even mixtures with low but above threshold concentrations of these odorants are likely to generate complaints.

Adult↗

PubMed2006

Fluctuations in diffusion processes in microgravity.

It has been shown recently that diffusion processes exhibit giant nonequilibrium fluctuations (NEFs). That is, the diffusing fronts display corrugations whose length scale ranges from the molecular to the macroscopic one. The amplitude of the NEF diverges following a power law behavior proportional to q(-4) (where q is the wave vector). However, fluctuations of wave number smaller than a critical "rolloff" wave vector are quenched by the presence of gravity. It is therefore expected that in microgravity conditions, the amplitude of the NEF should be boosted by the absence of the buoyancy-driven restoring force. This may affect any diffusion process performed in microgravity, such as the crystallization of a protein solution induced by the diffusion of a salt buffer. The aim of GRADFLEX (GRAdient-Driven FLuctuation EXperiment), a joint project of ESA and NASA, is to investigate the presence of NEFs arising in a diffusion process under microgravity conditions. The project consists of two experiments. One is carried out by UNIMI (University of Milan) and INFM (Istituto Nazionale per la Fisica della Materia) and is focused on NEF in a concentration diffusion process. The other experiment is performed by UCSB (University of California at Santa Barbara) concerning temperature NEF in a simple fluid. In the UNIMI part of the GRADFLEX experimental setup, NEFs are induced in a binary mixture by means of the Soret effect. The diagnostic method is an all-optical quantitative shadowgraph technique. The power spectrum of the induced NEFs is obtained by the processing of the shadowgraph images. A detailed description of the experimental apparatus as well as the ground-based experimental results is presented here for the UNIMI-INFM experiment. The GRADFLEX payload is scheduled to fly on the FOTON M3 capsule in April 2007.

Journal Article↗

PubMed2000

A fast method to sample real protein conformational space.

A fast computer program, FOLDTRAJ, to generate plausible random protein structures is reported. All-atom proteins are made directly in continuous three-dimensional space starting from primary sequence with an N to C directed build-up method. The method uses a novel pipelined residue addition approach in which the leading edge of the protein is constructed three residues at a time for optimal protein geometry, including the placement of cis proline. Build-up methods represent a classic N-body problem, expected to scale as N(2). When proteins become more collapsed, build-up methods are susceptible to backtracking problems which can scale exponentially with the number of residues required to back out of a trapped walk. We have provided solutions to both these problems, using a multiway binary tree that makes the N-body problem of bump-checking scale as NlogN, and speeding up backtracking by varying the number of tries before backtracking based on available conformational space. FOLDTRAJ is independent of energy potentials, other than that implicit in the geometrical properties derived by statistical studies of known structures, and in atomic Van der Waals radii. WHAT-CHECK shows that the program generates chirally and physically valid proteins with all bond lengths, angles and dihedrals within allowable tolerances. Random structures built using sequences from PDB files 1SEM, 2HPR, and 1RTP typically have 5-15% alpha-helical content (according to DSSP) and on the order of 20% beta-strand/extended content. Ensembles of random structures are compared with polymer theory and with experimentally determined fluorescence resonance energy transfer distances. Reasonably sized structure ensembles do sample most of the conformational space available to proteins. The method is also capable of protein reconstruction using Calpha--Calpha direction vectors, and it compares favorably with methods that reconstruct protein backbones based on alpha-carbon coordinates, having an average backbone and Cbeta root mean square deviation of 0.63 A for nine different protein folds. Proteins 2000;39:112-131.

Algorithms↗

PubMed1993

Direct in vivo gene transfer to airway epithelium employing adenovirus-polylysine-DNA complexes.

Adenovirus-polylysine-DNA complexes were evaluated for their capacity to accomplish direct in vivo gene transfer to airway epithelium employing a rodent model. Binary complexes containing transferrin or adenovirus, or combination complexes containing both transferrin and adenovirus, were evaluated. The highest in vitro gene transfer efficiency in primary cultures of airway epithelial cells was accomplished by the combination complexes. This result was paralleled in vivo. Transient gene expression of up to 1 week was observed with localization of the transduced cells to the region of the small airways. These results establish the feasibility of this type of approach for gene therapy applications.

Adenoviridae↗

PubMed1999

Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4.

A naturally occurring mutant of human thymidylate synthase (hTS) that contains a Tyr to His mutation at residue 33 was found to confer 4-fold resistance to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The crystal structure of hTS implicated this Tyr residue in a drug resistance mechanistic role that may include both substrate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287, 1995). Because of the existence of a defined kinetic scheme and the development of a bacterial expression vector for the overproduction of Escherichia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS. Nine mutant ecTS enzymes that differed in sequence at position 4 were generated. Mutants with a charged or polar side chain (Ser, Cys, Asp, and Arg) and Gly precipitated in the cell paste, resulting in no catalytic activity in cell-free extracts. Although most of the His 4 mutant precipitated, sufficient amounts remained in the cell-free extract to permit isolation to near homogeneity. Wild-type ecTS and mutants with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nearly 30% of the total cellular protein. The k(cat) values for the isolatable mutants were 2- to 10-fold lower than that of the wild-type enzyme, while the K(m) values for 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were similar for all the mutants. Dissociation constants for binary complex formation determined by stopped-flow spectroscopy were similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxythymidylate, indicating that this mutation does not significantly alter the binding of the natural nucleotide ligands. However, each mutant enzyme had three- to 5-fold lower affinity for FdUMP in the binary complex compared with the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP in the ternary complex. Analysis of k(burst) showed that the initial binding of CH(2)H(4)folate is weaker for each mutant compared to the wild-type enzyme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products.

Bacterial Proteins↗

PubMedDate not supplied

[Refined adenoviral transduction for controlled gene transfer into human adult mesenchymal stem cells].

AIM: Adenoviral gene transfer remains a powerful tool for basic research purposes. We hypothesize that adenoviral transduction of human mesenchymal stem cells (hMSC) in vitro can be improved by refined use of experimental parameters. METHODS: hMSCs were transduced by adenoviral vectors encoding Luciferase or BMP-2 at a selection of multiplicities of infection (MOI) and exposure times. Transgene production and total protein content were measured. To determine practical relevance, expression of the bone marker genes Runx2 and Type I collagen was analyzed by quantitative PCR. As a phenotypic marker alkaline phosphatase was assessed. ANOVA and post hoc statistical analyses were used to determine differences among data (p < 0.05). RESULTS: Prolonged exposure led to a decrease in transgene production and total protein content. Increasing MOI at exposure of up to 4 hours resulted in a higher production of the transgene. Transfer of the hBMP-2 gene promoted an enhanced lineage progression to the osteoblast phenotype indicating biological activity. CONCLUSION: Time of exposure is of major importance for toxicity in vitro and should not exceed 4 hours for hMSC. While increase in exposure time leads to cell death, surviving cells, up to a certain limit, seem to compensate by increasing production of the transgene indicating that transduction efficiency cannot be positively measured in a binary yes-or-no scheme.

Adenoviridae↗

PubMed2006

Eucalyptus.

Using Eucalyptus camaldulensis as a model system, we describe here a basic Agrobacterium-mediated genetic transformation protocol through organogenesis for the production of transgenic plants. Hypocotyl segments or cotyledon pieces from in vitro seedlings are used as starting materials. The explants are inoculated and cocultivated with a disarmed, binary strain of A. tumefaciens CIB542 harboring a mini Ti plasmid, pBI121. A modified Gamborg's B5 medium is used as the basal culture medium throughout stages of co-cultivation, callus induction and shoot regeneration. The incorporation of neomycin phosphotransferase II (nptII) and beta-glucuronidase (gus) genes into the plant nuclear genome are primarily verified by histochemical analysis and polymerase chain reaction (PCR). Modifications of this protocol to use in mature tissues derived from elite trees and other Eucalyptus species are also described.

Acclimatization↗

PubMed2005

Relaxation in a glassy binary mixture: comparison of the mode-coupling theory to a Brownian dynamics simulation.

We solved the mode-coupling equations for the Kob-Andersen binary mixture using structure factors calculated from Brownian dynamics simulations of the same system. We found, as was previously observed, that the mode-coupling temperature T(c) inferred from simulations is about two times greater than that predicted by the theory. However, we find that many time-dependent quantities agree reasonably well with the predictions of the mode-coupling theory if they are compared at the same reduced temperature epsilon = (T - T(c))/T(c), and if epsilon is not too small. Specifically, the simulation results for the incoherent intermediate scattering function, the mean square displacement, the relaxation time, and the self-diffusion coefficient agree reasonably well with the predictions of the mode-coupling theory. We find that there are substantial differences for the non-Gaussian parameter. At small reduced temperatures the probabilities of the logarithm of single particle displacements demonstrate that there is hopping-like motion present in the simulations, and this motion is not predicted by the mode-coupling theory. The wave-vector-dependent relaxation time is shown to be qualitatively different from the predictions of the mode-coupling theory for temperatures where hopping-like motion is present.

Journal Article↗

PubMed2004

Molecular dynamics simulations of acetonitrile/dimethyl sulfoxide liquid mixtures.

Binary liquid mixtures of dimethyl sulfoxide and acetonitrile at the three molar fractions 0.25, 0.50, and 0.75 have been investigated by molecular dynamics computer simulations. Thermodynamic states corresponding to liquid-vapor coexistence at a temperature of 298 K were considered. Intermolecular interactions were described by potential models of the site-site (12-6) Lennard-Jones plus Coulomb type that have been developed for the description of the pure liquids. Dimethyl sulfoxide has been represented by four interactions sites and acetonitrile by a three- as well as a six-site potential model. We have calculated thermodynamic properties and the intermolecular pair distribution functions. Intermolecular interaction energies indicate deviations from the behavior of ideal mixtures. The local mole fraction analysis demonstrates that dimethyl sulfoxide is preferentially solvated by acetonitrile and that the first solvation shell surrounding acetonitrile molecules is significantly enriched by dimethyl sulfoxide. The nonideal behavior in the mixtures is not affected by the choice of the three- or the six-site potential model for acetonitrile. Orientational correlations of dipole vectors within the first solvation shells indicate that the relative molecular orientations found in pure acetonitrile and dimethyl sulfoxide are maintained in the mixtures. Parallel and antiparallel dipole-dipole configurations determine first shell acetonitrile-dimethyl sulfoxide configurations. Dynamical features of the mixtures are discussed in terms of diffusion constants and orientational correlation times as obtained from the time correlation functions for linear velocities and molecular dipole moments, respectively. Computed relaxation times indicate faster reorientational motion for dimethyl sulfoxide if acetonitrile is added. In contrast, the orientational dynamics of acetonitrile becomes stronger correlated upon dilution with dimethyl sulfoxide. The diffusion coefficients for both compounds follow this tendency.

Journal Article↗

PubMed2002

Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method.

The efficiency of soybean [Glycine max (L.) Merrill] transformation was significantly increased from an average of 0.7% to 16.4% by combining strategies to enhance Agrobacterium tumefaciens-mediated T-DNA delivery into cotyledonary-node cells with the development of a rapid, efficient selection protocol based on hygromycin B. Wounded cotyledonary-node explants were inoculated with A. tumefaciens carrying either a standard-binary or super-binary plasmid and co-cultivated in the presence of mixtures of the thiol compounds, L-cysteine, dithiothreitol, and sodium thiosulfate. Transformed shoots began elongating only 8 weeks after co-cultivation. Southern analysis confirmed integration of the T-DNA into genomic DNA and revealed no correlation between the complexity of the integration pattern and thiol treatment applied at co-cultivation. All T(0) plants were fertile and the majority of the lines transmitted the beta-glucuronidase (GUS) phenotype in 3:1 or 15:1 ratios to their progenies.

Agrobacterium tumefaciens↗

PubMed2001

Non-oxidative mechanisms are responsible for the induction of mutagenesis by reduction of Cr(VI) with cysteine: role of ternary DNA adducts in Cr(III)-dependent mutagenesis.

Intracellular reduction of carcinogenic Cr(VI) generates Cr-DNA adducts formed through the coordination of Cr(III) to DNA phosphates (phosphotriester-type adduct). Here, we examined the role of Cr(III)-DNA adducts in mutagenesis induced by metabolism of Cr(VI) with cysteine. Reduction of Cr(VI) caused a strong oxidation of 2', 7'-dichlorofluoroscin (DCFH) and extensive Cr-DNA binding but no DNA breakage. Cr-DNA adducts induced unwinding of supercoiled plasmids and structural distortions in the DNA helix as detected by decreased ethidium bromide binding. Propagation of Cr-treated pSP189 plasmids in human fibroblasts led to a dose-dependent formation of the supF mutants and inhibition of replication. Blocking of Cr(III)-DNA binding by occupation of DNA phosphates with Mg(2+) or by sequestration of Cr(III) by inorganic phosphate or EDTA eliminated mutagenic responses and restored a normal yield of replicated plasmids. Dissociation of Cr(III) from DNA by a phosphate-based reversal procedure returned mutation frequency to background levels. The mutagenic responses at the different phases of the reduction reaction were unrelated to the amount of reduced Cr(VI) but reflected the number and the spectrum of Cr(III)-DNA adducts that were formed. Ternary cysteine-Cr(III)-DNA adducts were approximately 4-5 times more mutagenic than binary Cr(III)-DNA adducts. Although intermediate reaction products (CrV/IV, thiyl radicals) were capable of oxidizing DCFH, they were insufficiently reactive to damage DNA. Single-base substitutions at G/C pairs were the predominant type of Cr-induced mutations. The majority of mutations occurred at the sites where G had adjacent purine in the 3' or 5' position. Overall, our results present the first evidence that Cr(III)-DNA adducts play the dominant role in the mutagenicity caused by the metabolism of Cr(VI) by a biological reducing agent.

Base Sequence↗

PubMed1994

Long-term episomal gene delivery in human lymphoid cells using human and avian adenoviral-assisted transfection.

Human B-lymphoblastoid cells transformed by Epstein-Barr virus (EBV), a common source of established human cell lines, are generally refractory to standard DNA transfection methods. We report here the use of human and chicken adenoviruses for the stable delivery of plasmid DNA carrying the latent origin of replication, oriP, from EBV into human B-lymphoblastoid cells. Long-term hygromycin-resistant transformants of human lymphoblastoid cells belonging to the genetically unstable inherited disease Fanconi's anemia were obtained by adenoviral-assisted transfection of plasmid DNA complexed to either human adenovirus type 5 (Ad5) or avian adenovirus type 1 (CELO). Successful long-term transformation of human lymphoblastoid cells was unrelated to the efficiency of short-term transfection as measured by the transient expression level of a reporter lacZ gene. The human or avian adenoviral binary conjugates, with no other cell ligand, were sufficient for stable cell transformation. The amount of DNA established in the human B-cell transformants was approximately 100-fold higher with the CELO virus. We conclude that Ad5-assisted transfection is more suitable for transient gene expression studies in human lymphoblastoid cells, while CELO-assisted transfection appears the method of choice for stable genetic transformation of such cells. Such differential short- and long-term gene delivery strategy with Ad5 and CELO may be useful in gene therapy of human B cells involving cancers and inherited diseases, respectively.

Adenoviruses, Human↗