Development of animal models for adeno-associated virus site-specific integration

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.     

Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells

The levels and subcellular distribution of the interferon-inducible double-stranded RNA-dependent protein kinase PKR have been measured in human Daudi cells and stably transfected mouse NIH 3T3 cells expressing the human protein kinase. Immunofluorescence of intact cells and quantitative immunoblotting of cell extracts indicate that PKR occurs in both the cytoplasm and the cell nucleus, with staining specifically in the nucleolus. The ratio of cytoplasmic to nuclear PKR is approximately 5:1 in control cells; in response to interferon treatment the protein kinase is induced severalfold in the cytoplasm whereas the level in the nucleus does not increase significantly. Analysis of individual transfected cells by confocal microscopy reveals a pattern of distribution of PKR similar to that in Daudi cells, with immunostaining of cytoplasm and nucleoli. Similar results are observed whether cells expressing wild-type PKR or a catalytically inactive mutant form of the kinase are analyzed, but untransfected 3T3 cells are not stained by the antibody used. Two-dimensional isoelectric focusing analysis of PKR in whole cell extracts reveals the presence of multiple forms with different pI values whereas similar analysis of the nuclear fraction indicates only one predominant species with a relatively basic pI. These results suggest that PKR may have a role in the cell nucleus as well as the cytoplasm and that the subcellular distribution of the protein kinase may be related to post-translational modifications.     

Determination of deoxyribonucleoside triphosphate pool sizes in ribonucleotide reductase cDNA transfected human KB cells

Ribonucleotide reductase (RR) is a rate-limiting enzyme in DNA synthesis, which is responsible for controlling deoxyribonucleoside triphosphate (dNTP) pool size. It has been shown that transfection of RR M2 cDNA in human KB cells (M2-D clone) results in overexpression for the M2 subunit and resistance to hydroxyurea (HU). In this study, dNTP pool assays were performed to measure the pool sizes in six cell lines: two controls, three transfectants, and drug-induced HU-resistant (HUR) cells. Total dNTP levels among the six cell lines rose in the following order: KB wild-type, KB vector-only transfectant, M1 cDNA transfectant, M2 cDNA transfectant, M1/M2 cDNA transfectant, and HU-induced resistant clone. The dCTP levels of the cells mimicked the total dNTP pools on a smaller scale. The significant increases in the dCTP pool sizes of the M2-D, X-D, and HUR clones were proportional to their respective increases in RR activity. Relative to all other transfectants, the M1-D clone demonstrated lower dCTP levels but increased dATP pools. The M1-D clone demonstrated a significant resistance to dNTP inhibition of RR activity compared with the control KB wild-type cells. In contrast, a profound inhibition of dCTP and a decreased sensitivity to dATP inhibition was observed in M2-D, X-D, and HUR clones. In summary, M2 cDNA transfectants and HUR clones had increased RR activity as well as expanded dNTP pools, particularly dCTP, when compared with wild-type KB cells. These data provide evidence for the intertwined relationship between RR activity and dNTP pools.     

The neuronal alpha6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells

OBJECTIVE: Our purpose was to create a model for predicting amnionitis and rapid delivery in preterm labor patients by use of amniotic fluid interleukin-6 and clinical parameters. STUDY DESIGN: Amniotic fluid was cultured and analyzed, and a clinical score (incorporating gestational age, amniotic fluid Gram stain, glucose, leukocyte esterase, and maternal serum C-reactive protein) was determined in 111 patients diagnosed with preterm labor. Statistical analysis involved t tests, chi2, logarithmic regression, and multivariate regression analysis (P < or = .05). RESULTS: The incidence of positive amniotic fluid cultures was 8.7% (9 of 103 patients). Patients with positive cultures of the amniotic fluid had a shorter delivery interval (4.8 +/- 7.5 vs 28.9 +/- 25.4 days, P < .001). Patients with elevated amniotic fluid interleukin-6 (> or = 7586 pg/ml) were more likely to have a positive amniotic fluid culture (relative risk = 8.8, 95% confidence interval = 1.6 to 47.4, P < .001) and to be delivered within 2 days (relative risk = 16.8, 95% confidence interval = 4.5 to 62.7, P < .001). Stepwise multivariate regression analysis yielded a model using interleukin-6, cervical dilatation, and gestational age (r2 = 0.63, P < .001) with a specificity of 100% for predicting delivery within 2 days of amniocentesis. CONCLUSIONS: A mathematical model using maternal amniotic fluid interleukin-6 seems to be a useful clinical tool for quantifying the interval to preterm birth for patients in preterm labor.     

Intracellular metabolism of human apolipoprotein(a) in stably transfected Hep G2 cells

Lipoprotein(a) [Lp(a)] consists of LDL and the glycoprotein apolipoprotein(a) [apo(a)], which are covalently linked via a single disulfide bridge. The formation of Lp(a) occurs extracellularly, but an intracellular assembly in human liver cells has also been claimed. The human apo(a) gene locus is highly polymorphic due to a variable number of tandemly arranged kringle IV repeats. The size of apo(a) isoforms correlates inversely with Lp(a) plasma concentrations, which is believed to reflect different synthesis rates. To examine this association at the cellular level, we analyzed the subcellular localization and fate of apo(a) in stably transfected HepG2 cells. Our results demonstrate that apo(a) is synthesized as a precursor with a lower molecular mass which is processed into the mature, secreted form. The retention times of the precursor in the ER positively correlated with the sizes of apo(a) isoforms. The mature form was observed intracellularly at low levels and only in the Golgi apparatus. No apo(a) was found to be associated with the plasma membrane. Under temperature-blocking conditions, we did not detect any apo(a)/apoB-100 complexes within cells. This finding was confirmed in HepG2 cells transiently expressing KDEL-tagged apo(a). The precursor and the mature forms of apo(a) were found in the ER and Golgi fractions, respectively, also in human liver tissue. From our data, we conclude that in HepG2 cells the apo(a) precursor, dependent on the apo(a) isoform, is retained in the ER for a prolonged period of time, possibly due to an extensive maturation process of this large protein. The assembly of Lp(a) takes place exclusively extracellularly following the separate secretion of apo(a) and apoB.     

Magnetic selection of transiently transfected cells

An efficient and rapid method for selecting transiently transfected cells is described. A plasmid encoding for a neural cell-specific surface marker is co-transfected into mammalian cells along with the gene of interest. After uptake and expression of these two plasmids, the transfected cells are immuno-adsorbed to magnetic beads pre-coated with antibodies against the surface marker. These immuno-complexes are then isolated by means of a strong magnet. In a single round of magnetic selection, we were able to enrich the cell population more than 7-fold for a co-transfected reporter. These specifically selected cells can now be used for either further cultivation or for immediate analysis. This method has been shown to be effective on HeLa and on CV-1 cells and is expected to give similar results on any other transfectable, non-neuronal cell lines.     

Assembly of hair keratins in transfected epithelial cells

To define the interactions required for the filament assembly of differentiation-specific keratins, active copies of mouse hair keratin mHa1 and mHb4 genes were introduced into a rat kangaroo kidney epithelial cell line (PtK2) and a rat stratified squamous epithelial cell line (rat epidermal keratinocyte). In PtK2 transient transfectants, when introduced individually or in combination, mHa1 and mHb4 formed aggregates of ring-like structures of various sizes at the perinuclear region with no evidence of organization into a keratin network. These aggregates altered the distribution of the endogenous keratins and vimentin. In most of the cells carrying the ring-like structures of mHa1 and mHb4 around the nucleus, the endogenous keratin network collapsed and localized around the nucleus. Furthermore, the densely accumulated endogenous keratin surrounded the ring-like aggregates with partial co-localization. However, when transfected into the rat epidermal keratinocytes, mHa1 and mHb4 were able to co-localize with the well-developed cytoskeleton of endogenous keratins. These results showed that, in contrast to keratin pairs K5/K14 and K8/K18, the mHa1/mHb4 pair is unable to develop an extensive keratin network on its own and that there are possible differential abilities among these hair keratins and other keratins to form well-developed cytoplasmic networks.     

Study of human foamy virus proviral integration in chronically infected murine cells

BACKGROUND: An altered nocturnal sleep pattern and a dysfunction of the hypothalamic-pituitary-adrenocortical system are neurobiological abnormalities typical for depression. A persistence of these neurobiological alterations during remission has been shown to be associated with an increased risk for a relapse. However, it remains unclear whether these persisting abnormalities are trait markers indicative of an increased vulnerability for affective disorders or only represent 'biological scars' acquired during past episodes. Thus, respective examinations need to be performed in the premorbid state in order to answer this open question. METHODS: In the present article we have summarized the various results of the index investigation of a prospectively designed study in which we investigated 54 healthy first-degree relatives (high-risk probands; HRPs) of patients with an affective disorder using polysomnography, the combined dexamethasone corticotropine-releasing hormone (DEX-CRH) test and psychometric measurements. RESULTS: In the cross-sectional part of this study the HRPs, as a group, exhibited a 'depression-like' sleep EEG profile and DEX-CRH test result, while their psychometric profile was characterized by elevated scores on the measures 'Rigidity' and 'Autonomic lability'. On an individual level, 35% of the HRPs were identified as conspicuous in at least two of the three areas under investigation. CONCLUSIONS: The question of whether these abnormalities do indeed reflect trait markers indicative of an increased vulnerability for depression will be answered by the longitudinal part of the study that allows for the retrospective identification of the premorbid status of those HRPs who develop an affective disorder during the follow-up period.     

Molecular analysis of mutations induced by acrolein in human fibroblast cells using supF shuttle vector plasmids

Types of mutations induced by acrolein in the supF gene on the shuttle vector plasmid pMY189 replicated in normal human fibroblast cells were examined. Base sequence analysis of 92 plasmids with mutations in the supF gene revealed that the majority of the mutations were base substitutions (76%) and the others were deletions and insertions (24%). Single base substitutions were most frequently found (46%), while multiple base substitutions were 18% and tandem (two adjacent) base substitutions were 12% of the mutations. Of the base substitution mutations, G:C to T:A transversions were 44% and G:C to A:T transitions were 24%. The mutations were distributed not randomly but located at several hotspots. Acrolein produced DNA intra-strand cross-links between guanine residues, which might be responsible for rather high induction of the tandem base substitution mutations.     

Subcellular distribution and turnover of presenilins in transfected cells

The mechanisms by which mutations in presenilin-1 (PS1) and presenilin-2 (PS2) result in the Alzheimer's disease phenotype are unclear. Full-length PS1 and PS2 are each processed into stable proteolytic fragments after their biosynthesis in transfected cells. PS1 and PS2 have been localized by immunocytochemistry to the endoplasmic reticulum (ER) and Golgi compartments, but previous studies could not differentiate between the full-length presenilin proteins and their fragments. We carried out subcellular fractionation of cells stably transfected with PS1 or PS2 to determine the localization of full-length presenilins and their fragments. Full-length PS1 and PS2 were principally distributed in ER fractions, whereas the N- and C-terminal fragments were localized predominantly to the Golgi fractions. In cells expressing the PS1 mutant lacking exon 9 (DeltaE9), we observed only full-length molecules that were present in the ER and Golgi fractions. The turnover rate was considerably slower for the DeltaE9 holoprotein, apparently due to decreased degradation within the ER. Our results suggest that that full-length presenilin proteins are primarily ER resident molecules and undergo endoproteolysis within the ER. The fragments are subsequently transported to the Golgi compartment, where their turnover rate is much slower than that of the full-length presenilin in the ER.     

Multiplication of adeno-associated virus type 1 in cells coinfected with a temperature-sensitive mutant of human adenovirus type 31

The maturation of the stato-acoustic nerve in the cat was studied by light and electron microscopy from the fetal stage to the adult. Measurement of the outer diameter of the fibers and the study of the myelination process revealed that myelination begins earlier for the vestibular nerve than for the cochlear nerve: by the fifty-third day of gestation 64% of the vestibular fibres have already passed the promyelin stage whereas for the cochlear nerve this promyelin stage begins for the majority of fibers on the fifty-seventh gestation day. Afterward, maturation proceeds more rapidly for the cochlear nerve. In the case of both nerves, maturation is still incomplete at two months of age. Concerning the relationship between the thickness of the myelin sheath and the axoplasmic diameter, there is already a good correlation by the fifty-seventh day of gestation in the vestibular nerve, whereas it appears several days after birth in the cochlear nerve.     

Role of the proteasome complex in degradation of human CYP2E1 in transfected HepG2 cells

The aim of the present study was to characterize human CYP2E1 turnover and examine the possible role of the proteasome proteolytic pathway in the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Microsomes isolated from MVh2E1-9 cells catalyzed a slow degradation of the expressed CYP2E1, which was prevented by the addition of 4-methylpyrazole, a ligand which stabilizes CYP2E1. The addition of the cytosolic fraction of the HepG2 cells to the microsomes produced rapid degradation of CYP2E1. This rapid degradation required MgATP and was completely prevented by 4-methylpyrazole. Pulse-chase experiments after labeling CYP2E1 with [35S]-methionine and immunoprecipitation with anti-human CYP2E1 IgG indicated a biphasic turnover of CYP2E1 with half-lives of 2.5 and 6 hours. The addition of Czb-Ile-Glu(OtBu)-Ala-Leucinal(PSI) as a cell penetrating proteasome inhibitor, at concentrations ranging from 5 to 80 microM resulted in protection against the degradation of CYP2E1. PSI also increased the steady state accumulation of CYP2E1, consistent with its inhibition of CYP2E1 turnover. These results suggest that the proteasome complex plays a major role in the degradation of human CYP2E1 in the transfected HepG2 cells.     

Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers

Starburst polyamidoamine (PAMAM) dendrimers are a new type of synthetic polymer characterized by a branched spherical shape and a high density surface charge. We have investigated the ability of these dendrimers to function as an effective delivery system for antisense oligonucleotides and 'antisense expression plasmids' for the targeted modulation of gene expression. Dendrimers bind to various forms of nucleic acids on the basis of electrostatic interactions, and the ability of DNA-dendrimer complexes to transfer oligonucleotides and plasmid DNA to mediate antisense inhibition was assessed in an in vitro cell culture system. Cell lines that permanently express luciferase gene were developed using dendrimer mediated transfection. Transfections of antisense oligonucleotides or antisense cDNA plasmids into these cell lines using dendrimers resulted in a specific and dose dependent inhibition of luciferase expression. This inhibition caused approximately 25-50% reduction of baseline luciferase activity. Binding of the phosphodiester oligonucleotides to dendrimers also extended their intracellular survival. While dendrimers were not cytotoxic at the concentrations effective for DNA transfer, some non-specific suppression of luciferase expression was observed. Our results indicate that Starburst dendrimers can be effective carriers for the introduction of regulatory nucleic acids and facilitate the suppression of the specific gene expression.     

Alpha 5 subunit forms functional alpha 3 beta 4 alpha 5 nAChRs in transfected human cells

nAChRs heterologously expressed in human cells after transient transfection with alpha 3 beta 4 alpha 5 or alpha 3 beta 4 subunit cDNAs exhibited similar sensitivities to antagonists and comparable functional channel profiles. However, the sum of two Hill equations was required for best fitting the ACh dose-current response curves after co-expression of alpha 5, alpha 3 and beta 4 subunits. One component was comparable to that obtained in alpha 3 beta 4-transfected cells, while the additional component, putatively attributed to an alpha 3 beta 4 alpha 5 nAChR population, showed a Hill coefficient > 2 and a nine-fold greater half-maximal ACh concentration (EC50). These results suggest that the alpha 5 subunit participates in the assembly of alpha 3 beta 4 alpha 5 nAChRs complexes in human cells, adding a new member to the family of neuronal nicotinic receptors.     

Extended life of human corneal endothelial cells transfected with the SV40 large T antigen

PURPOSE: To transfect human corneal endothelial cells with a plasmid vector coding for the SV40 large T antigen to extend the life of the cells in culture. METHODS: Human corneal endothelial cells were transfected with the SV40 large T antigen-coding plasmid pSV3neo using the electroporation method. Transfected and control cells were propagated in culture until senescence. Polymerase chain reaction and immunofluorescence were used to demonstrate messenger RNA and protein, respectively, for the Simian virus 40 large T antigen in the transfected cells. Polymerase chain reaction and hot blotting were used to demonstrate messenger RNA coding for several growth factors and receptors in transfected and control cells. RESULTS: The transfected cells continued to proliferate to 38 passages (more than 120 population doublings) in culture (control cells, 8 population doublings). Transfected cells, but not control cells, expressed messenger RNA coding for the Simian virus 40 large T antigen. Similarly, immunofluorescent staining with monoclonal antibodies demonstrated that the Simian virus 40 large T antigen protein was present in the nucleus of the transfected cells. Transfected cells were shown to produce messenger RNA coding for epidermal growth factor, epidermal growth factor receptor, basic fibroblast growth factor, fibroblast growth factor receptor-1, interleukin-1 alpha, the interleukin-1 receptor, transforming growth factor beta-1, and the glucocorticoid receptor. Qualitative expression of the messenger RNA coding for each of these modulators was similar in proliferating primary corneal endothelial cells and proliferating or confluent transfected corneal endothelial cells. CONCLUSIONS: In culture, the life of human corneal endothelial cells transfected with a plasmid vector coding for the Simian virus 40 large T antigen is extended. This study suggests that human corneal endothelial cells have the capacity for extensive proliferation, but the proliferation of untransfected cells is regulated through mechanisms that have not yet been characterized.     

Detection and quantification of apoptosis in transiently transfected adherent cells

Apoptosis is a highly conserved form of cell death present in all eukaryotic cell types and controlled by multiple genes. Several methods have been developed to quantify apoptosis, but none is adapted for all cell types. It is particularly difficult to reliably assay apoptosis of adherent cells. We describe a new, rapid and reliable flow cytometric method which can be used for quantifiying apoptosis in a sub-population of transiently transfected adherent cells. This technique is based on the detection of transfected cells and the apoptotic sub-population by immunofluorescence and Annexin-V labelling, respectively.     

Targeted replacement of normal and mutant CFTR sequences in human airway epithelial cells using DNA fragments

Recent studies have reported that mutant genomic cystic fibrosis (CF) transmembrane conductance regulator ( CFTR ) sequences can be corrected in transformed CF airway epithelial cell lines by targeted replacement with small fragments of DNA with wild-type sequence. To determine if the observed genotype modification following small fragment homologous replacement (SFHR) was limited to transformed CF cell lines, further studies were carried out in both transformed and non-transformed primary normal airway epithelial cells. The endogenous genotype of these normal cell lines was modified following liposome or dendrimer transfection using DNA fragments with DeltaF508 CFTR sequence (488 nt, complementary single strands) designed to also contain a unique restriction enzyme cleavage site (Xho I). Replacement at the appropriate genomic locus by exogenous DeltaF508 CFTR DNA and its expression as mRNA was demonstrated by PCR amplification of genomic DNA and mRNA-derived cDNA as well as Xho I digestion of the PCR products. These studies show that SFHR occurs in both transformed and non-transformed primary human airway epithelial cells and indicate that single base substitution (the silent mutation giving rise to the Xho I site) and deletion or insertion of at least three consecutive bases can be achieved in both normal and CF epithelial cells. Furthermore, these studies reiterate the potential of SFHR as a strategy for a number of gene targeting applications, such as site-specific mutagenesis, development of transgenic animals, development of isogenic cell lines and for gene therapy.     

Spatial integration in human smooth pursuit

When viewing a moving object, details may appear blurred if the object's motion is not compensated for by the eyes. Smooth pursuit is a voluntary eye movement that is used to stabilize a moving object. Most studies of smooth pursuit have used small, foveal targets as stimuli (e.g. Lisberger SG and Westbrook LE. J Neurosci 1985;5:1662-1673.). However, in the laboratory, smooth pursuit is poorer when a small object is tracked across a background, presumably due to a conflict between the primitive optokinetic reflex and smooth pursuit. Functionally, this could occur if the motion signal arising from the target and its surroundings were averaged, resulting in a smaller net motion signal. We asked if the smooth pursuit system could spatially summate coherent motion, i.e. if its response would improve when motion in the peripheral retina was in the same direction as motion in the fovea. Observers tracked random-dot cinematograms (RDC) which were devoid of consistent position cues to isolate the motion response. Either the height or the density of the display was systematically varied. Eye speed at the end of the open-loop period was greater for cinematograms than for a single spot. In addition, eye acceleration increased and latency decreased as the size of the aperture increased. Changes in the density produced similar but smaller effects on both acceleration and latency. The improved pursuit for larger motion stimuli suggests that neuronal mechanisms subserving smooth pursuit spatially average motion information to obtain a stronger motion signal.     

Invasion of fibrosarcoma cells transfected with PAI-2 gene

The effect of PAI-2 on the invasion of fibrosarcoma cells in vitro and in vivo was investigated. The control cells (C+, C+ pem) and PAI-2 transfectants (C+ exp) were used in the assay of the degradation of isotopically labelld 3H-ECM and were injected sc into athymic/nude mice. Recombinant PAI-2 could inhibit efficiently the degradation of 3H-ECM by tumor cells (86.5%). The PAI-2 transfectants remained tumorigenic in nude mice, but tumors originating from the PAI-2 transfectants showed histologically the presence of a thick capsule which was absent in tumors from control cells.