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At least 433 records · Page 24Linked to original sources

PubMed1993

Comparative abilities of athymic nude mice and severe combined immune deficient (SCID) mice to accept transplants of induced rat mammary carcinomas: enhanced transplantation efficiency of those rat mammary carcinomas that have elevated expression of neu oncogene.

Grafts of primary carcinogen (DMBA)-induced mammary carcinomas from Sprague-Dawley rats have a poor transplantation efficiency in athymic nude mice. Further compromising these mice immunologically via whole-body irradiation and/or splenectomy, or the administration of hormonal growth factors (estrogen and progesterone) to these mice, did not significantly alter transplantation efficiency. Use of strains of mice that are more immune-impaired than the athymic nude mouse, i.e., the athymic nude-beige-XID mouse (T-cell and LAK-cell deficient) or mice with severe combined immune deficiency (SCID) (which lack functional T cells and B cells) also failed to improve transplantation efficiency. In contrast, transplantation efficiency was sharply increased when primary neu-induced rat mammary carcinomas from female Sprague-Dawley rats were used. These mammary carcinomas, unlike the DMBA-induced rat mammary carcinomas, have a very high level of expression of neu; transplantation of these tumors to either athymic nude mice or SCID mice was considerably more efficient. Thus, these data provide evidence that enhanced expression of neu confers heightened efficiency in the transplantation of primary rat mammary carcinomas to immune-deficient mice (athymic-nude or SCID). Increased neu expression was a greater determinant than more compromised immune states in the transplantation of these rat mammary carcinomas. This biological characteristic of neu expression in mammary carcinomas provides new, additional insight into the importance of this oncogene in mammary tumorigenic processes and may explain, at least in part, the reported inverse relationship between human breast carcinoma neu expression and patient prognosis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1994

RNA 3' end signals of the S.pombe ura4 gene comprise a site determining and efficiency element.

We have defined sequences in the 3' non-coding region of the Schizosaccharomyces pombe ura4 gene that are required for efficient mRNA 3' end formation. Three separate sequence elements have been identified. Two of these are site determining elements which specify alternative sites of polyadenylation [the major poly(A) site and a minor downstream poly(A) site]. The third sequence, located downstream of both poly(A) sites, functions as an efficiency element that enhances utilization of either polyadenylation site. By employing sensitive RT-PCR analysis, we demonstrate that although low levels of transcripts are detected up to the efficiency element, none is detected beyond this point. The downstream site determining element and efficiency element have both been delineated to specific 16 nt sequences which we show are together sufficient for ura4 mRNA 3' end formation. We have further characterized the interaction between these two elements and show that the efficiency element behaves in a position-independent, orientation-dependent manner, but cannot form 3' ends independently of the site determining element. Surprisingly, we find that the efficiency element can be functionally replaced by a second copy of either site determining element. We present a model for the mechanism of RNA 3' end formation of the ura4 gene and note that this bipartite structure for a poly(A) signal in S.pombe may be related to the AAUAAA and downstream GU-rich sequences of poly(A) signals in mammalian genes.

Base Sequence↗

PubMed1995

Efficiency of spermatogenesis.

Spermatogenesis is a process of division and differentiation by which spermatozoa are produced in seminiferous tubules. A measure of efficiency of spermatogenesis is the estimated number of spermatozoa produced per day per gram of testicular parenchyma. This measure is not influenced by species differences in testicular size; however, it is influenced by species differences in the numerical density of germ cells and in the life spans of these cells. Seminiferous tubules are composed of somatic cells (myoid cells and Sertoli cells), and germ cells (spermatogonia, spermatocytes, and spermatids). Activity of these three germ cells divide spermatogenesis into spermatocytogenesis, meiosis, and spermiogenesis, respectively. Spermatocytegenesis involves mitotic cell division to increase the yield of spermatogenesis and to produce stem cells and primary spermatocytes. Meiosis involves duplication and exchange of genetic material and two cell divisions that reduce the chromosome number and yield four spermatids. Spermiogenesis is the differentiation of spherical spermatids into mature spermatids which are released at the luminal free surface as spermatozoa. The spermatogenic cycle is superimposed on the three major divisions of spermatogenesis. Spermatogenesis and germ cell degeneration can be quantified from numbers of germ cells in various steps of development throughout spermatogenesis, and quantitative measures are related to number of spermatozoa in the ejaculate. Germ cell degeneration occurs throughout spermatogenesis; however, the greatest impact occurs during spermatocytogenesis and meiosis. Efficiency of spermatogenesis is related to the amount of germ cell degeneration, pubertal development, season of the year, and aging of humans and animals. Number of Sertoli cells and amount of smooth endoplasmic reticulum of Leydig cells (but not Leydig cell number) are related to efficiency of spermatogenesis. In humans, efficiency of spermatogenesis is reflected in number of spermatogenic stages per cross-section and number of missing generations within each stage; however, the arrangement of stages along the tubular length does not reflect differences in the efficiency of spermatogenesis. In short, spermatogenesis involves both mitotic and meiotic cell divisions and an unsurpassed example of cell differentiation in the production of the spermatozoon, and daily sperm production per g parenchyma is a measure of its efficiency.

Aging↗

PubMedDate not supplied

High efficiency lentiviral gene delivery in non-dividing cells by deoxynucleoside treatment.

BACKGROUND: Gene therapy has recently been advanced by the development of HIV-based vectors that are able to transduce some non-dividing cells. The manipulation of most non-dividing cells remains, however, scarcely efficient. One of the biological mechanisms postulated to prevent powerful transduction of quiescent cells by lentiviral vectors is the paucity of deoxynucleotides (dNTPs). In this study, a novel delivery strategy is developed to improve significantly the efficiency of HIV-based vectors in transducing non-dividing cells. This approach is based on increasing the intracellular availability of dNTPs by incubating target cells with the dNTP precursors, deoxynucleosides (dNSs). METHODS: Mature human monocyte-derived macrophages (14-21 days old) were transduced at a low multiplicity of infection (MOI) of HIV vectors carrying a reporter gene. dNSs were added to the medium during transduction (5 mM dNS) and immediately before post-transduction culture (2.5 mM dNS). Macrophages were harvested 2-7 days after transduction and assayed for transgene expression by cytofluorimetry. RESULTS: The addition of dNS to the medium significantly enhanced the efficiency of transduction of human macrophages by HIV-based vectors. The percentage of cells expressing the transgene rose up to 50% in the presence of dNS, increasing the basal transduction levels up to 35-fold (average=10.8-fold). Furthermore, treatment with dNTP precursors compensated for the wide inter-donor variability, allowing the highest enhancement effects in donors with the lowest basal transduction efficiencies. CONCLUSIONS: This is the first demonstration that a single treatment of non-dividing target cells with exogenous dNS can enhance the efficiency of lentiviral-mediated transduction of cells, allowing for high efficiency gene transfer. The effects of dNTP precursors compensated for both the poor basal levels and the wide inter-donor variability, two major limitations for the transduction of non-dividing cells. Macrophages are a representative model of cells whose permissiveness to gene delivery was increased up to levels suitable for genetic manipulation applications. This simple approach might be transferred to a broader range of quiescent cell types that are scarcely susceptible to lentiviral-based gene delivery due to low dNTP levels.

3T3 Cells↗

PubMed2003

Uptake of plasmid/glycosylated polymer complexes and gene transfer efficiency in differentiated airway epithelial cells.

BACKGROUND: We have studied gene transfer efficiency of glycosylated polylysines and glycosylated polyethylenimines as vectors in immortalized differentiated airway gland serous cells and primary cultures of human airway surface epithelial cells. METHODS AND RESULTS: In both cell types, lactosylated PEI was more efficient for gene transfer than unsubstituted PEI and lactosylated polylysine which requires the presence of endosomolytic agents. However, for all the vectors tested, gene transfer efficiency was lower in differentiated cells as compared with poorly differentiated cells. The presence of membrane lectins, i.e. cell surface sugar-specific receptors, was evaluated using fluorescein-conjugated neoglycoproteins and microscopy or flow cytometry. In differentiated airway surface epithelial cells, membrane lectins were not expressed and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were not incorporated. This accounted in part for the lack of gene transfer efficiency in these cells. In contrast, in differentiated airway gland serous cells, expression of lectins and their endocytotic properties appeared to be similar to that observed in undifferentiated cells, and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were incorporated in similar amounts by cells in both differentiated states CONCLUSIONS: Glycosylated PEI appears to be a promising gene delivery system since it is more efficient than the sugar-free polymer and does not require endosomolytic agents. However, in differentiated airway gland serous cells, a low gene transfer efficiency was observed that could not be attributed to low expression of membrane lectins or low uptake of glycosylated complexes. An impaired intracellular trafficking of glycosylated complexes in differentiated airway gland serous cells is suggested.

Cell Line↗

PubMed2004

Efficient transfection of non-proliferating human airway epithelial cells with a synthetic vector system.

BACKGROUND: The poor transfection efficiency of most synthetic vectors in non-dividing, airway epithelial cells is limiting the development of gene therapy for respiratory diseases such as cystic fibrosis. METHODS: Cultured airway epithelial cells, quiescent or replicating, were transfected with a lipid/peptide synthetic vector system and transfection efficiency assessed by luciferase assays or flow cytometry for green fluorescent protein expression. Tight junctions were disrupted by treatment with ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA). Cell division was inhibited by aphidicolin and assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation and assay by fluorescence microscopy or flow cytometry. In vivo transfection was performed by intratracheal instillation. RESULTS: Initial studies showed that this synthetic vector transfected 8% of non-dividing epithelial cells in culture compared with 45% of subconfluent, dividing cells. However, formulation of the vector with EGTA, which disrupts cellular tight junctions, induced a four-fold increase in the transfection efficiency of confluent, non-mitotic cells. The EGTA enhancement of transfection also correlated with a four-fold increase in vector binding to the cells, presumably via exposed basolateral receptors. Flow cytometry and double immunofluorescent staining for reporter gene expression and BrdU incorporation indicated efficient transfection of non-proliferating cells in a confluent monolayer by this vector. Furthermore, in vivo transfection of murine lung by intratracheal administration showed a six-fold enhancement of luciferase expression when lungs were pre-treated with EGTA. CONCLUSIONS: This study confirms the hypothesis that a lipid/peptide vector transfects non-dividing cells with high efficiency which contributes to its previously reported efficiency in transfecting bronchial epithelium, making it a good choice for gene transfer studies in the lung.

Egtazic Acid↗

PubMed2004

Dependence of the cellular internalization and transfection efficiency on the structure and physicochemical properties of cationic detergent/DNA/liposomes.

BACKGROUND: Control of the structure and physicochemical properties of DNA complexed with nonviral vectors is essential for efficient biodistribution and gene delivery to cells. Cationic liposomes interact with DNA giving transfection competent but large and heterogeneous aggregates. On the other hand, cationic detergents condense DNA into small homogeneous but reversible complexes inefficient for transfection. METHODS: In order to combine the favorable features of both vectors, ternary complexes were prepared by adding cationic liposomes to plasmid DNA condensed by cationic detergents. The structure and physicochemical properties of these complexes were investigated by electron microscopy, quasi-elastic light scattering, gel electrophoresis and fluorescence techniques. These data were then correlated with the transfection efficiency and intracellular trafficking of the ternary complexes determined by luciferase gene expression and confocal microscopy, respectively. RESULTS: The ternary complexes were found to form small, homogeneous, globular, stable and positively charged particles with a highly dense and packed lamellar internal structure differing from the multilamellar structure (L(alpha)(C)) of the corresponding lipoplexes. In the presence of serum, the ternary complexes were more efficiently internalized into cells, less toxic and showed 20-fold higher transfection efficiency than lipoplexes. CONCLUSIONS: This study showed that small, monodisperse and highly stable complexes could be obtained by precompaction of DNA with cetyltrimethylammonium bromide, followed by addition of cationic lipids. The higher efficiency of the ternary complexes with respect to their corresponding lipoplexes was related to their internal structure which prevents their dissociation by serum proteins and allows efficient internalization in the target cells.

Animals↗

PubMed2006

In vivo estimation of the flow-driven adiabatic inversion efficiency for continuous arterial spin labeling: a method using phase contrast magnetic resonance angiography.

The accurate quantification of perfusion with arterial spin labeling (ASL) requires consideration of a number of factors, including the efficiency of the inversion and control pulses used for spin labeling. In this study the effects of spin velocity on continuous ASL efficiency when using the amplitude modulated control strategy were investigated using simulations of the Bloch equations. The inversion efficiency was determined in vivo by combining the simulations with phase-contrast velocity mapping data acquired at the level of the tagging plane. Using this novel method, an average inversion efficiency of 69% was calculated for a group of 28 subjects, in good agreement with experimental data reported previously. There was, however, a large range in inversion efficiency measured across the subject group (50-76%), indicating that the velocity dependence of the amplitude modulated control efficiency may introduce additional variability into the perfusion calculations if not properly taken into account.

Algorithms↗

PubMed1992

Efficient secretion in yeast based on fragments from K1 killer preprotoxin.

The alpha and beta components of the secreted K1 killer toxin of Saccharomyces cerevisiae are derived from residues 45-147 and 234-316, respectively, of the 316 residue preprotoxin (ppTox). The beta N-terminus is produced by Kex2 cleavage after Lys Arg233; when beta la (the mature sequence of beta-lactamase) is fused at this site and the fusion is expressed from the PGK promoter in pDT17, a multicopy plasmid, unexpectedly modest levels of beta la secretion resulted. Over-expression of Kex2 failed to increase beta la secretion while a kex2-null mutation reduced secretion by 98%. beta la secretion in a Kex+ strain was not enhanced by inactivation of the alpha toxin component or by deletion of most of its central hydrophobic segments. However SP-beta la, produced by deletion of ppTox residues 35-176, expressed 10-fold higher beta la activity and the precursor was now secreted with similar efficiency in a kex2-null strain. Fusions of beta la to ppTox at Ala34 or Ala46 also led to efficient secretion in both KEX2 and kex2-null strains. Since these beta la fusions differ only in segments well downstream of the signal peptide and all had similar transcript levels, the efficiency of beta la secretion is apparently determined by the efficiency with which these fusions are translocated to the Golgi compartment where Kex2 is active. Efficiency is high for the shorter fusions, but is 10% or less for the longer fusions; even this fraction is apparently diverted to the vacuole if not cleaved by Kex2. SP-beta la was the most efficient construct tested; secreted beta la reached 4% of total cell protein, modestly exceeding levels produced by fusion to the MF alpha 1-encoded prepro alpha-factor, suggesting potential for the production of foreign proteins in yeast.

Amino Acid Sequence↗

PubMed2002

Quantitative immunoblot assay for assessment of liposomal antibody conjugation efficiency.

Routine direct assessment of immunoglobulin (Ig)-liposome(lp) conjugation efficiency has been impeded by phospholipid interference with standard protein and immunoassay methods. Rabbit IgG conjugated to anionic liposomes was quantitated in immunoblots using computer image analysis techniques. Lp-coupled Ig was separated from free Ig by dialysis in disposable Spectra/Por units (MWCO 300 kDa). Differential Lowry protein assay (DLA) of the thiolated Ig reactant and the dialyzate provided an estimate of conjugation efficiency that was compared to the results of the immunoblot assay (IBA). The color response of Ig-lp in the IBA was about an order of magnitude greater than rabbit IgG alone, requiring the synthesis of an Ig-lp standard in which the Ig conjugation efficiency was assessed by radiotracer methodology. The use of the same standard in three colorimetric protein assays verified the accuracy of the IBA and demonstrated that the colorimetric assays could be employed to determine Ig-lp conjugation efficiency. In terms of sensitivity and specificity, however, the IBA is better suited for routine assessment of laboratory-scale Ig-lp conjugation efficiencies. The DLA was found to be an unsatisfactory measure of conjugation efficiencies because an interfering substance was apparently released by Ig-lp preparations.

Animals↗

PubMed1997

Efficient in vivo gene transfer into the heart in the rat myocardial infarction model using the HVJ (Hemagglutinating Virus of Japan)--liposome method.

The lack of efficient treatment for myocardial infarction remains an unresolved problem in the field of cardiovascular disease. Gene therapy may be a potential therapeutic strategy for the treatment of myocardial infarction. However, current methods of in vivo gene transfer into the heart are limited by their low efficiency and/or potential toxicity. In the present study, we developed an efficient technique of gene transfer into the intact heart in vivo using the Sendai virus (HVJ: Hemagglutinating Virus of Japan)--liposome method. We used the beta-galactosidase gene, luciferase gene and human angiotensin converting enzyme (ACE) gene as markers. In vivo gene transfer into the rat heart was performed as follows: (1) direct injection into the rat heart, (2) incubation within the pericardium, and (3) infusion into a coronary artery. Direct injection of the HVJ-liposome complex containing the beta-galactosidase vector into the rat heart resulted in limited staining of beta-galactosidase 3 days after transfection. To compare transfection efficiency between "naked" plasmid DNA transfection and the HVJ-liposome method, we also transfected the luciferase reporter gene into the heart. Luciferase activity was significantly higher in hearts transfected by the HVJ-liposome method than that in hearts transfected by direct "naked" plasmid transfection (P < 0.01). To confirm the successful gene in the protein level, we measured ACE activity in the hearts. Cardiac ACE activity was significantly increased in hearts transfected with human ACE gene as compared to hearts transfected with control vector (P < 0.01). On the other hand, incubation of HVJ-liposome complex, containing beta-galactosidase vector, within the pericardium resulted in widespread staining of cardiac myocytes and fibroblasts, mainly located in several surface layers beneath the pericardium. More importantly, widespread stained areas of beta-galactosidase were also observed in the middle of the myocardium around the vasa vasorum. We also examined the efficiency of gene transfer by the HVJ-liposome method in a rat myocardial infarction model. In the infarction model, using the pericardium incubation approach, staining for beta-galactosidase was observed in the viable cells around the infarction area. Finally, direct infusion of the HVJ complex, containing the beta-galactosidase vector, into coronary artery also resulted in widespread staining of beta-galactosidase in cardiac myocytes around the microvasculature. Using direct injection, we found significant injury to the myocardium and severe fibrosis at the injection site, whereas no apparent injury was observed using pericardium incubation and coronary infusion. There was no evidence of cytotoxicity or inflammation caused by the HVJ-liposome complex itself. Overall, we have established an efficient in vivo gene transfer method into the heart using the HVJ-liposome method. Direct infusion into the coronary artery resulted in widespread transfection without damaging the myocytes; incubation within the pericardium demonstrated the usefulness of the HVJ-liposome method for studying cardiac function and as a means of gene therapy for cardiovascular diseases.

Animals↗

PubMed1999

A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes.

Human immunodeficiency virus (HIV, lentivirus) type-1 based vectors have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and long term transgene expression. We used a three-plasmid expression system to generate pseudotyped lentivirus-based vectors by transient transfection of human embryonic kidney 293T cells in the presence of sodium butyrate, which is known to activate the long terminal repeat-directed expression of HIV. Using this system we successfully generated versatile high titer lentivirus at titers of up to 2 x 10(8) transducing units/ml (TU/ml), and improved transduction efficiency in various cell types from seven to over twenty fold. We demonstrate its applicability of these vectors for the efficient transduction of non-dividing cells, including post mitotic beating rat cardiac myocytes and well-differentiated rat L6 myofibers. While both lentivirus-based and murine retrovirus-based vectors effectively transduced dividing cardiac fibroblasts and L6 muscle myoblasts in culture, lentivirus-based vectors also efficiently transduced cardiac myocytes and yielded titers of (6.3 +/- 1.2) x 10(5) TU/ml; however murine retrovirus-based vectors showed low transduction efficiency with titers reaching only (8.9 +/- 2.1) x 10(2) TU/ml. Furthermore, even 12 days after induction of differentiation of L6 myofibers, lentivirus-mediated transduction of beta-galactosidase (beta-Gal) at approximately 30-40% of the maximum expression levels achieved in replicating myoblasts. In contrast, the expression of beta-Gal following transduction of the myofibers by murine retrovirus-based vectors fell to less than 1% of an already reduced level of transduction in undifferentiated confluent myoblasts. These results demonstrate that lentivirus-based vectors can efficiently transduce both well-differentiated cardiac myocytes and differentiated myofibers. This appears to be an efficient method and provides a new tool for research and therapy for cardiovascular diseases.

Animals↗

PubMed2002

Improvement in adenoviral gene transfer efficiency after preincubation at +37 degrees C in vitro and in vivo.

Adenovirus is a widely used vector in gene transfer experiments because it produces high transduction efficiency in vitro and in vivo by means of the coxsackie-adenovirus receptor (CAR) and major histocompatibility complex (MHC) class I alpha-2 domain. Adenoviral gene transfer efficiency has been reported to correlate with cellular CAR expression. We report here a simple method to increase adenoviral gene transfer efficiency in cells that do not express high levels of CAR: preincubation of adenovirus for 30-40 minutes at +37 degrees C significantly increased the transduction efficiency in vitro in CHO and BALB/3T3 cells, in which CAR is expressed at very low levels. Increased transduction efficiency of preincubated adenovirus was also detected in vivo in rat brain tissue. In addition, we found that adenoviruses were rapidly inactivated in human serum in a complement-independent manner, whereas fetal bovine serum (FBS) had hardly any effects on the viral infectivity. We conclude that preincubation of adenoviral vectors at +37 degrees C may substantially increase gene transfer efficiency in applications in which target cells do not express high levels of CAR.

3T3 Cells↗

PubMed1985

Mechanical efficiency of the left ventricle as a function of preload, afterload, and contractility.

We have recently shown that the mechanical efficiency of the contractile machinery of the canine left ventricle is constant at 30%-50%, independent of its loading, heart rate, and inotropic conditions. In contrast, the conventional mechanical efficiency of the ventricle is known to vary between 0 and 30%, depending on these conditions. In this study, we derived an equation for the conventional mechanical efficiency as a function of ventricular preload, afterload, and contractility, based on the constant mechanical efficiency of the contractile machinery. In deriving this equation, we fully utilized our new concept of the total mechanical energy of the left ventricle, i.e., systolic pressure-volume area, and our recent findings of the linear relationship between left ventricular oxygen consumption and the systolic pressure-volume area as well as the dependence of this relation on the ventricular inotropic state. As a result, the conventional mechanical efficiency of the left ventricle was found to change between 0 and 25% as an explicit function of these cardiodynamic and inotropic conditions. Using this function, we obtained combinations of loading and inotropic conditions to maximize the conventional mechanical efficiency of the left ventricle.

Animals↗

PubMed1995

Evaluation of right ventricular function by assessment of cardiac efficiency: influence of induction of anaesthesia in coronary artery bypass grafting patients.

Considering the heart as a physical pump cardiac efficiency is calculated from the ratio of cardiac work performed to the maximum level of energy of the heart. The aim of the study was to compare cardiac efficiency with cardiac output and right ventricular ejection fraction. Nine patients scheduled for coronary artery bypass grafting were investigated. A femoral arterial and a right ventricular ejection fraction pulmonary artery catheter were placed in the awake state. Anaesthesia was induced with eltanolone and fentanyl. Cardiac output, pulmonary artery and central venous pressures, and right ventricular ejection fraction were measured in the awake state (baseline), 2 min after induction of anaesthesia and 1 and 5 min after intubation. Cardiac efficiency was calculated by dividing the stroke work by the maximum energy of the heart as calculated from the pressure volume diagram. An analysis of variance was carried out for cardiac efficiency, cardiac output and right ventricular ejection fraction. Cardiac efficiency was significantly (p < 0.05) reduced 1 min after intubation from 28 +/- 11 to 14 +/- 5%. In contrast the right ventricular ejection fraction (from 48 +/- 10 to 35 +/- 13%) and cardiac output (from 6.5 +/- 1.5 to 5.3 +/- 1.2 L/min) did not change significantly during the induction of anaesthesia. Cardiac efficiency was found to be a more sensitive parameter to describe changes in the right ventricular function than the ejection fraction and cardiac output during induction of anaesthesia with eltanolone and fentanyl which was used as a model to vary cardiac performance and afterload.

Aged↗

PubMed1991

Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice.

We are studying the tissue-specific expression of the sheep milk-whey protein gene, beta-lactoglobulin. We have used sequences derived from this gene to target the expression of biomedical proteins into milk with the intention to exploit this technology in transgenic sheep as a means of protein production. In the present study, a series of beta-lactoglobulin hybrid genes and beta-lactoglobulin minigenes were evaluated for expression in the mammary gland of transgenic mice. In particular, we have assessed whether there is a requirement for introns for efficient transgene expression in the mammary gland, since the coding sequences of many candidate proteins are available only as cDNAs. The results suggest that the inclusion of natural introns in constructs can enhance the efficiency of transgene expression. Thus, a hybrid construct comprising 4.3 kb of the immediate 5' flanking sequences of beta-lactoglobulin fused to a genomic minigene encoding human alpha-antitrypsin (alpha 1AT) was expressed much more efficiently than an alpha 1AT-cDNA construct containing the same beta-lactoglobulin segment. Similarly, the intact beta-lactoglobulin gene was expressed more efficiently than the corresponding intronless beta-lactoglobulin minigene. This effect was not seen in transient expression experiments in baby hamster kidney cells when beta-lactoglobulin-alpha 1AT constructs were driven by SV40 enhancer sequences. The effect cannot be explained by a simple requirement for splicing, since the inclusion of the first beta-lactoglobulin intron into cDNA constructs encoding human alpha 1AT or beta-lactoglobulin itself failed to enhance the efficiency of transgene expression. It is concluded that sequence elements within introns may interact with the upstream 5' flanking sequences of beta-lactoglobulin and enable the latter to function efficiently in the mammary gland of transgenic mice.

Animals↗

PubMed1999

Study of gene delivery in a rabbit vein graft model. Improvement of the efficiency of gene transfer into vein grafts.

Gene therapy is a therapeutic strategy in treating cardiovascular disease. Vein graft failure, the major limitation on coronary artery bypass surgery, may be amenable to gene approaches. Some studies describe gene therapies using functioning genes to prevent vein graft stenosis. Gene transfer efficiency remains a major issue. In this rabbit vein graft model, we studied gene delivery using a replication-deficit recombinant adenovirus to improve gene transfer efficiency into vein grafts. The adenovirus vector that contains the E.coli lacZ gene encoding beta gal was used because this vector is widely used and thought to be effective. Gene transfer was detected by X-gal staining. We hypothesized that dimethylsulfoxide and hyaluronidase, both drug delivery enhancers, would improve efficiency and that, in transfer to adventitia, direct injection would be more effective than dwelling. We studied 3 gene delivery methods to intima and media (controls, using dimethylsulfoxide and using hyaluronidase before transfection) and 2 delivery methods to adventitia (direct injection and dwelling). We used 6 rabbits per delivery method. X-gal stained positive cell rates were counted using light microscopy. Our findings indicate that (1) dimethylsulfoxide increased the efficiency of transfection to media and intima, (2) hyaluronidase increased intimal transfection efficiency, (3) direct injection to adventitia was more effective than dwelling. These findings suggest that in vein grafting, our methods are feasible for improving gene transfer efficiency.

Adenoviridae↗

PubMed2000

Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies.

The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene6, and compared it with two other transfection reagents, Tfx-50 and LipoTaxi. For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVbeta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGene6 is a highly efficient transfection reagent that may be useful for in vitro non-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery.

Adult↗