Β-gal Expression Research Articles

Identification of a Site of Hsp27 Binding with Hsp27 and αB-Crystallin as Indicated by the Yeast Two-Hybrid System

The small heat-shock proteins (sHsp), including Hsp27 and αB-crystallin, usually form large oligomers in cells. It has been suggested that the sHsp form oligomers by binding either a conserved C-terminal amino acid sequence or the less conserved N-terminal region. However, the site of binding has not been precisely determined. We used the yeast two-hybrid system to investigate binding of full-length rat Hsp27 or fragments of Hsp27 to full-length rat Hsp27 or αB-crystallin molecules. A series of cDNAs coding for fragments of Hsp27 were generated and ligated with the coding sequence for the binding domain of the yeast Gal4p transcription factor. These cDNAs were each transfected into yeast that had been transfected to express full-length rat Hsp27 or αB-crystallin fused with the DNA binding domain of Gal4p. Yeast cells transfected with both plasmids were assayed, by both a β-galactosidase (β-gal) filter assay and a quantitative liquid assay, for activation of Gal4p-driven β-gal expression. Results indicated that the N-terminal domain of Hsp27 consisting of amino acids 1-124 did not bind to Hsp27 or αB-crystallin. The predominant Hsp27-Hsp27/αB-crystallin binding domain was the conserved C-terminal region consisting of amino acids 141-206, particularly amino acids 141-176.

Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinant adeno-associated virus vectors.

The delivery of recombinant genes to cardiomyocytes holds promise for the treatment of a variety of cardiovascular diseases. Previous gene transfer approaches that used direct injection of plasmid DNA or replication-defective adenovirus vectors have been limited by low transduction frequencies and transient transgene expression due to immune responses, respectively. In this report, we have tested the feasibility of using intramyocardial injection or intracoronary infusions of recombinant adeno-associated virus (rAAV) vectors to program transgene expression in murine cardiomyocytes in vivo. We constructed an rAAV containing the LacZ gene under the transcriptional control of the cytomegalovirus (CMV) promoter (AAVCMV-LacZ). We then injected 1x10(8) infectious units (IU) of this virus into the left ventricular myocardium of adult CD-1 mice. Control hearts were injected with the AdCMV-LacZ adenovirus vector. Hearts harvested 2, 4, and 8 weeks after AAVCMV-LacZ injection demonstrated stable beta-galactosidase (beta-gal) expression in large numbers of cardiomyocytes without evidence of myocardial inflammation or myocyte necrosis. In contrast, the AdCMV-LacZ-injected hearts displayed transient beta-gal expression, which was undetectable by 4 weeks after injection. Explanted C57BL/6 mouse hearts were also perfused via the coronary arteries with 1.5x10(9) IU of AAVCMV-LacZ and assayed 2, 4, and 8 weeks later for beta-gal expression. beta-Gal expression was detected in <1% of cardiomyocytes at 2 weeks after perfusion but was detected in up to 50% of cardiomyocytes 4 to 8 weeks after perfusion. Direct intramyocardial injection or coronary artery perfusion with rAAV vectors can be used to program stable transgene expression in cardiomyocytes in vivo. rAAV appears to represent a useful vector for the delivery of therapeutic genes to the myocardium.

Gene Expression in Black Tiger Prawns Following Intramuscular Injection of β-gal Plasmid

Gene expression in black tiger prawns (Penaeus monodon) was studied following intra-muscular injection of CMVβGal plasmid into the second abdominal segment. We used an in situ staining technique to detect β-gal expression in one- and three-month-old injected prawns. We found that only one of the three-month-old prawns expressed the marker gene (2 days after injection), and the site of expression was confined to the sixth abdominal segment away from the injection site. We repeated the experiment on a new batch of three-month-old prawns, but using fluorometric determination technique. This time we found that β-gal expression was detected (6/42) at the site of injection after 2, 7, and 14 days. In two other test samples, transgene expression was detected in the sixth abdominal segment only, further confirming the possibility of injected DNA dispersal. The results of the study also suggest that direct gene transfer is a feasible technique in black tiger prawns.

Overexpression of TTF-1 and PAX-8 restores thyroglobulin gene promoter activity in ARO and WRO cell lines

Background: In anticipation of developing gene therapy against thyroid carcinoma we created an expression vector using the thyroglobulin (Tg) gene promoter. The inhibition of both Tg and thyroid-specific transcription factor (TTF-1 and PAX-8) gene expression, however, has been well documented in thyroid carcinomas. We therefore examined the effects of overexpression of TTF-1 and PAX-8 on Tg gene promoter activity in the human thyroid carcinoma cell lines, ARO (anaplastic) and WRO (follicular). Methods: ARO, WRO, and nonthyroid cells were transfected with an expression vector in which β-galactosidase (β-gal) is driven by the Tg gene promoter (β-gal). Tg, TTF-1, and PAX-8 gene expression were also examined by reverse transcriptase–polymerase chain reaction (RT-PCR). Results: ARO and WRO exhibited decreased gene expression of Tg, TTF-1, and PAX-8. Transfection with TG-—gal alone exhibited minimal β-gal expression, whereas cotransfection with TTF-1 and PAX-8 resulted in markedly increased expression. There was no evidence of β-gal expression with or without TTF-1 and PAX-8 in nonthyroid cells. Conclusions: Weak Tg gene promoter activity in ARO and WRO is associated with decreased expression of transcription factors TTF-1 and PAX-8 but can be restored with their overexpression. This model may serve as a template on which to further develop cell-specific gene therapy against thyroid carcinoma. (Surgery 1998;124:1100-5.)

Gene Transfer in Dendritic Cells, Induced by Oral DNA Vaccination With Salmonella typhimurium, Results in Protective Immunity Against a Murine Fibrosarcoma

AbstractA live attenuated AroA− auxotrophic mutant ofSalmonella typhimurium (SL7207) has been used as carrier for the pCMVβ vector that contains the β-galactosidase (β-gal) gene under the control of the immediate early promoter ofCytomegalovirus (CMV). We tested whether orally administered bacterial carrier could enter and deliver the transgene to antigen-presenting cells (APCs) through the natural enteric route of infection and whether β-gal expression could generate a protective response against an aggressive murine fibrosarcoma transduced with the β-gal gene (F1.A11) that behaves operationally as a tumor-associated antigen. After three courses, at 15-day intervals, mice developed both cell-mediated and systemic humoral responses to β-gal. Mice vaccinated with the Salmonella harboring pCMVβ, but not with plasmid-less carrier, showed resistance to a challenge with F1.A11 cells. These experiments suggest that Salmonella-based DNA immunization allows us to specifically target antigen expression in vivo to APCs. To prove that the transgene is actually expressed by APCs as a function of an eukaryotic promoter, the green fluorescent protein (GFP) was placed under the control of either the eukariotic CMV or a prokaryotic promoter. Using cytofluorometric analysis, GFP was detected only in splenocytes of mice receiving a Salmonella carrier harboring GFP under the CMV promoter. These results indicate that transgene expression occurs because of a Salmonella-mediated gene transfer to eukaryotic cells. Finally, approximately 19% of the splenocytes expressed GFP. Among them, F4/80+ macrophages and CD11cbright dendritic cells (DCs) were scored as positive for GFP expression. Extensive work has been performed trying to optimize the way to transfect DCs, ex vivo, with genes coding for relevant antigens. We show here, for the first time, that DCs can be directly and specifically transduced in vivo such to induce DNA vaccination against tumors.© 1998 by The American Society of Hematology.

Gene Transfer in Dendritic Cells, Induced by Oral DNA Vaccination With Salmonellatyphimurium, Results in Protective Immunity Against a Murine Fibrosarcoma

A live attenuated AroA− auxotrophic mutant ofSalmonella typhimurium (SL7207) has been used as carrier for the pCMVβ vector that contains the β-galactosidase (β-gal) gene under the control of the immediate early promoter ofCytomegalovirus (CMV). We tested whether orally administered bacterial carrier could enter and deliver the transgene to antigen-presenting cells (APCs) through the natural enteric route of infection and whether β-gal expression could generate a protective response against an aggressive murine fibrosarcoma transduced with the β-gal gene (F1.A11) that behaves operationally as a tumor-associated antigen. After three courses, at 15-day intervals, mice developed both cell-mediated and systemic humoral responses to β-gal. Mice vaccinated with the Salmonella harboring pCMVβ, but not with plasmid-less carrier, showed resistance to a challenge with F1.A11 cells. These experiments suggest that Salmonella-based DNA immunization allows us to specifically target antigen expression in vivo to APCs. To prove that the transgene is actually expressed by APCs as a function of an eukaryotic promoter, the green fluorescent protein (GFP) was placed under the control of either the eukariotic CMV or a prokaryotic promoter. Using cytofluorometric analysis, GFP was detected only in splenocytes of mice receiving a Salmonella carrier harboring GFP under the CMV promoter. These results indicate that transgene expression occurs because of a Salmonella-mediated gene transfer to eukaryotic cells. Finally, approximately 19% of the splenocytes expressed GFP. Among them, F4/80+ macrophages and CD11cbright dendritic cells (DCs) were scored as positive for GFP expression. Extensive work has been performed trying to optimize the way to transfect DCs, ex vivo, with genes coding for relevant antigens. We show here, for the first time, that DCs can be directly and specifically transduced in vivo such to induce DNA vaccination against tumors.© 1998 by The American Society of Hematology.

Maternal Nanos regulates zygotic gene expression in germline progenitors of Drosophila melanogaster

Maternal Nanos (Nos) protein is required for germline development in Drosophila embryos. Here we show that Nos regulates zygotic gene expression in the germline progenitors, or pole cells. In order to probe the gene expression in pole cells, we screened ten enhancer-trap lines which showed β-gal expression in pole cells. All of these enhancer-trap markers were fully activated in pole cells after their migration to the embryonic gonads. In the pole cells lacking Nos, the expression of nine out of ten enhancer-trap markers was affected. Among nine markers, five (Type-A) were prematurely expressed in the pole cells during the course of their migration. The expression of other four markers (Type-B) initiated correctly after pole-cell migration, but their expression was significantly reduced. Thus, we conclude that the maternal Nos plays a dual role in zygotic gene regulation in pole cells: to define the stages of expression for Type-A markers, and to enhance expression for Type-B markers. Contrary to our results, Heller and Steinmann-Zwicky (1998)have recently reported that no premature expression of Type-A markers occurs in the pole cells of embryos derived from nos mutant females. This discrepancy is due to the difference in the nos mutant alleles used for these analyses. We used the much stronger allele, nos BN .

Promyelinating Schwann Cells Express Tst-1/SCIP/Oct-6

Tst-1/SCIP/Oct-6, a POU domain transcription factor, is transiently expressed by developing Schwann cells and is required for their normal development into a myelinating phenotype. In tst-1/scip/oct-6-null sciatic nerves, Schwann cells are transiently arrested at the "promyelinating" stage, when they have a one-to-one relationship with an axon but before they have elaborated a myelin sheath. To determine when Schwann cells express Tst-1/SCIP/Oct-6, we examined beta-galactosidase (beta-gal) expression in heterozygous tst-1/scip/oct-6 mice, in which one copy of the tst-1/scip/oct-6 gene has been replaced with the LacZ gene. beta-Gal expression from the LacZ gene seems to parallel Tst-1/SCIP/Oct-6 expression from the endogenous tst-1/scip/oct-6 gene in developing and regenerating sciatic nerves. Furthermore, electron microscopic examination of 5bromo-4-chloro-3-indolyl-beta-D-galactopyranoside- (X-gal) and halogenated indolyl-beta-D-galactoside- (Bluo-gal) stained nerves showed that promyelinating Schwann cells express the highest levels of beta-gal, both in developing and in regenerating nerves. Thus, the expression of beta-gal, a surrogate marker of Tst-1/SCIP/Oct-6, peaks at the same stage of Schwann cell development at which development is arrested in tst-1/scip/oct-6-null mice, indicating that Tst-1/SCIP/Oct-6 has a critical role in promyelinating Schwann cells.

In vivo gene transfer to the mouse oviduct epithelium

Objective: To transfect the mouse oviduct in vivo. Design: Prospective study. Setting: Research laboratory. Animal(s): Sixteen female Swiss albino mice. Intervention(s): The left oviduct of 10 female mice was instilled with a liposome DNA solution. In addition, 2 control mice received liposome solution, 2 received phosphate-buffered saline solution, and 2 received no injection. Main Outcome Measure(s): The expression of the gene transfected (β-galactosidase) was detected in the oviduct epithelium with the use of a routine histochemical analysis. Result(s): The 90% of the female mice that were transfected with liposome/β-Gal complexes expressed the gene in the oviduct mucosa. The controls did not show β-Gal expression. Conclusion(s): Cationic liposome/DNA complexes can be used for in vivo transfection of the mouse fallopian tubes. The foreign gene expression occurs in clusters of cells located along the mucosa of the isthmus and juncture regions.

Efficient Gene Transfer into Cardiac Myocytes Using Adeno-Associated Virus (AAV) Vectors

Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containingβ-galactosidase (β-gal) genein vitro, and the expression ofβ-gal in CM was evaluated by X-gal staining andβ-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and theβ-gal expression increased to 31.1±4.6 ng/mg protein in a MOI-dependent manner (MOI: 104to 106particles/cell). Theβ-gal expression was also increased in an incubation period-dependent manner (1–24 h).β-gal expression was maximal at day 3 and then gradually decreased with time. However,β-gal expression at day 14 was almost the same level as that at day 1 (45.5±5.9v55.2±6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZex vivo. When theβ-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.

Evidence that the RdeA protein is a component of a multistep phosphorelay modulating rate of development in Dictyostelium.

We have isolated an insertional mutant of Dictyostelium discoideum that aggregated rapidly and formed spores and stalk cells within 14 h of development instead of the normal 24 h. We have shown by parasexual genetics that the insertion is in the rdeA locus and have cloned the gene. It encodes a predicted 28 kDa protein (RdeA) that is enriched in charged residues and is very hydrophilic. Constructs with the DNA for the c-Myc epitope or for the green fluorescent protein indicate that RdeA is not compartmentalized. RdeA displays homology around a histidine residue at amino acid 65 with members of the H2 module family of phosphotransferases that participate in multistep phosphoryl relays. Replacement of this histidine rendered the protein inactive. The mutant is complemented by transformation with the Ypd1 gene of Saccharomyces cerevisiae, itself an H2 module protein. We propose that RdeA is part of a multistep phosphorelay system that modulates the rate of development.

Growth and ?-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis

Growth and β-galactosidase (β-gal) expression were characterized in the yeast Kluyveromyces lactis strain NRRL Y-1118 growing in aerobic chemostat cultures under carbon, nitrogen or phosphate limitation. In lactose or galactose-limited cultures, β-gal accumulated in amounts equivalent to 10-12% of the total cell protein. The induced β-gal expression was repressed when cells were grown under N- or P-limitation. In lactose medium, enzyme levels were 4-8 times lower than those expressed in C-limited cultures. A similar response was observed when galactose was the carbon source. These results suggest that a galactose-dependent signal (in addition to glucose) may have limited induction when cells were grown in carbon-sufficient cultures. Constitutive β-gal expression was highest in lactate-limited and lowest in glucose-limited media and was also repressed in glucose-sufficient cultures. Other K. lactis strains (NRRL Y-1140 and CBS 2360) also showed glucose repression (although with different sensitivity) under non-inducing conditions. We infer that these strains share a common mechanism of glucose repression independent of the induction pathway. The kinetics of β-gal induction observed in C-limited cultures confirms that β-gal induction is a short-term enzyme adaptation process. Applying a lactose pulse to a lactose-limited chemostat culture resulted in 'substrate-accelerated death'. Immediately after the pulse, growth was arrested and β-gal was progressively inactivated. Yeast metabolism in C-limited cultures was typically oxidative with the substrate being metabolized solely to biomass and CO 2 . Cells grown under P- or N-limitation, either with glucose or lactose, exhibited higher rates of sugar consumption than C-limited cells, accumulated intracellular reserve carbohydrates and secreted metabolic products derived from the glycolytic pathway, mainly glycerol and ethanol.

Selective disruption of genes transiently induced in differentiating mouse embryonic stem cells by using gene trap mutagenesis and site-specific recombination.

A strategy employing gene trap mutagenesis and site-specific recombination (Cre/loxP) has been used to identify genes that are transiently expressed during early mouse development. Embryonic stem cells expressing a reporter plasmid that codes for neomycin phosphotransferase and Escherichia coli LacZ were infected with a retroviral gene trap vector (U3Cre) carrying coding sequences for Cre recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the two selectable marker genes and consequently the expression of beta-galactosidase (beta-Gal). As a result, clones in which U3Cre had disrupted genes that were only transiently expressed could be selected. Moreover, U3Cre-activating cells acquired a cell autonomous marker that could be traced to cells and tissues of the developing embryo. Thus, when two of the clones with inducible U3Cre integrations were passaged in the germ line, they generated spatial patterns of beta-Gal expression.

−245 bp of 5′-Flanking Region From the Human Platelet Factor 4 Gene Is Sufficient to Drive Megakaryocyte-Specific Expression In Vivo

AbstractPlatelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5′-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with −245 bp of 5′-flanking region were more active than constructs with −2 kb of 5′-flanking region in vitro. We created two independent transgenic mouse lines with a −245-bp hPF4/lacZ construct. Cells from these mice were tested for β-galactosidase (β-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed β-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no β-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The −245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.

−245 bp of 5′-Flanking Region From the Human Platelet Factor 4 Gene Is Sufficient to Drive Megakaryocyte-Specific Expression In Vivo

Platelet factor 4 (PF4) serves as a lineage-specific marker of megakaryocyte development. We previously identified two positively acting sequences in the human platelet factor 4 (hPF4) gene promoter that synergized to drive high-level luciferase reporter gene expression in vitro. Using portions of the hPF4 5′-flanking region linked to the lacZ reporter gene, we observed in this investigation that constructs with −245 bp of 5′-flanking region were more active than constructs with −2 kb of 5′-flanking region in vitro. We created two independent transgenic mouse lines with a −245-bp hPF4/lacZ construct. Cells from these mice were tested for β-galactosidase (β-gal) expression at the mRNA level by Northern blot and semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and at the protein level by immunohistochemistry assay. Mice from one line showed β-gal expression specifically in all megakaryocytes of all ploidy classes from bone marrow and in platelets. Expression level was comparable to that driven by the 1.1-kb rat PF4 promoter in other transgenic mouse lines. Those in the second line showed no β-gal expression in megakaryocytes, platelets, or any of the eight organs tested. The −245-bp hPF4 promoter is capable of driving reporter gene expression in a megakaryocyte-specific manner in transgenic mice. The small size of this megakaryocyte-specific promoter is compatible with that required in some viral vectors and may provide a model for targeting gene expression to megakaryocytes.

DNA-polycation nanospheres as non-viral gene delivery vehicles

Nanospheres synthesized by salt-induced complex coacervation of cDNA and polycations such as gelatin and chitosan were evaluated as gene delivery vehicles. DNA-nanospheres in the size range of 200–750 nm could transfect a variety of cell lines. Although the transfection efficiency of the nanospheres was typically lower than that of lipofectamine and calcium phosphate controls in cell culture, the β-gal expression in muscle of BALB/c mice was higher and more sustained than that achieved by naked DNA and lipofectamine complexes. This gene delivery system has several attractive features: (1) ligands can be conjugated to the nanosphere for targeting or stimulating receptor-mediated endocytosis; (2) lysosomolytic agents can be incorporated to reduce degradation of the DNA in the endosomal and lysosomal compartments; (3) other bioactive agents or multiple plasmids can be co-encapsulated; (4) bioavailability of the DNA can be improved because of protection from serum nuclease degradation by the polymeric matrix; (5) the nanosphere can be lyophilized for storage without loss of bioactivity.

Liposome-Mediated Gene Transfer Into Established CNS Cell Lines, Primary Glial Cells, and In Vivo

Sufficient gene transfer into CNS-derived cells is the most crucial step to develop strategies for gene therapy. In this study liposome-mediated gene transfer using a β-galactosidase (β-GAL) reporter gene was performed in vitro (C6 glioma cells, NT2 neuronal precursor cells, 3T3 fibroblasts, primary glial cells) and in vivo. Using Trypan blue exclusion staining, optimal lipid concentration was observed in the range of 10–12 μg/mL. Under optimal conditions (80,000 cells/16 mm well, incubation overnight, lipid/DNA ratio = 1:18) a high transfection rate was achieved (<9% for C6 cells; <1% for NT2 cells). In primary cultures of glial cells a fair amount of positive stained cells (glial cell) was found, but the transfection efficiency was lower (<0.1%). A “boost-lipofection” markedly increased (twice) lipofection efficiency in C6 cells. Expression of β-GAL reached a maximum after 3–5 days. When the liposome–DNA complexes were injected/infused directly into the brains of adult rats, several weakly stained cells could be observed in the brain region adjacent to the injection site. It is concluded that liposome-mediated gene transfer is an efficient method for gene transfer into CNS cells in vitro, but the transfection efficiency into the rat brain in vivo is far too low and therefore not applicable.

Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle

We have developed protective interactive noncondensing (PINC) polymers, such as poly(N-vinyl pyrrolidone) (PVP) and poly(vinyl alcohol) (PVA), to protect plasmids from extracellular nuclease degradation while allowing the flexible complex to diffuse throughout the muscle tissue. Molecular modeling, zeta potential modulation, and ethidium bromide intercalation studies were performed to assess the mechanism of interaction between PVP and plasmid. The effect of salt concentration, pH, and polymer–plasmid ratios were investigated. We have correlated these variables with β-galactosidase (β-gal) expression after intramuscular administration to rats. PVP can form hydrogen bonds with the base pairs within the major groove of DNA at pH 4.0. The PVP–plasmid interaction results in a complex that is more hydrophobic (less negatively charged) than the uncomplexed plasmid due to the vinyl backbone of PVP. Up to a ten-fold enhancement in gene expression in rat muscle over the use of `naked' DNA has been demonstrated using these systems. A linear structure–activity relationship (SAR) was found between the percent vinyl pyrrolidone monomer content in poly(vinyl pyrrolidone-covinyl acetate) polymers and β-gal expression in muscle. Modulation of the interaction between PINC polymers and plasmid directly impacts the levels of gene expression in vivo. The linear SAR is being used to design novel PINC polymers with enhanced binding affinity to plasmids.

Gene Transfer Into the Donor Heart During Cold Preservation for Heart Transplantation

Background. Ex vivo gene transfer to heart grafts may hold promise as a means of changing alloreactivity or xenoreactivity after transplantation. However, it remains to be determined how effectively gene transfer can be accomplished within a short time in cold-stored grafts that are ready to be transplanted.Methods. We performed an experimental study using a replication-defective adenovirus (Adex1CALacZ) encoding the Escherichia coli β-galactosidase (β-gal) gene to perform gene transfer to heart grafts awaiting transplantation. Thirty hearts of Wistar rats were removed and their coronary arteries were perfused with University of Wisconsin solution containing 1 × 109, 1 × 1010, or 1 × 1011 plaque-forming units of the recombinant adenovirus at 4°C for 60 minutes. As a control, other hearts were perfused with University of Wisconsin solution with an adenoviral vector that did not contain the β-gal gene (Adex1w1) for the same period. After perfusion, the grafts were implanted in the necks of syngeneic adult rats. The grafts were removed each week after transplantation and their expression of β-gal was assessed by 5-bromo-4-chloro-3-indoyl-β-d-galactoside staining.Results. Successful gene transfer and expression of the β-gal gene were demonstrated in adenovirus-perfused hearts. Gene transfer occurred preferentially in the cardiomyocytes over the endothelial cells and smooth muscle cells of the coronary vessels. In hearts perfused with 1 × 109 plaque-forming units of the adenovirus, gene expression persisted for 4 weeks after transfer, but it diminished gradually and was minimal by day 28. Histologic analyses revealed slight inflammatory reactions in the myocardium. In hearts perfused with 1 × 1010 and 1 × 1011 plaque-forming units of the adenovirus, β-gal diminished 3 weeks after transplantation and a prominent infiltration of leukocytes was recognized in the myocardium.Conclusions. This study demonstrated that the cardiomyocytes of heart grafts express an exogenous gene product after adenovirus-mediated gene transfer under hypothermic preservation conditions. However, immune or inflammatory reactions to recombinant adenoviruses must be taken into account when a large number of adenoviruses are injected into the coronary arteries.

Liposome-mediated gene transfer into established CNS cell lines, primary glial cells, and in vivo.

Sufficient gene transfer into CNS-derived cells is the most crucial step to develop strategies for gene therapy. In this study liposome-mediated gene transfer using a beta-galactosidase (beta-GAL) reporter gene was performed in vitro (C6 glioma cells, NT2 neuronal precursor cells, 3T3 fibroblasts, primary glial cells) and in vivo. Using Trypan blue exclusion staining, optimal lipid concentration was observed in the range of 10-12 microg/mL. Under optimal conditions (80,000 cells/16 mm well, incubation overnight, lipid/DNA ratio = 1:18) a high transfection rate was achieved (<9% for C6 cells; <1% for NT2 cells). In primary cultures of glial cells a fair amount of positive stained cells (glial cell) was found, but the transfection efficiency was lower (<0.1%). A "boost-lipofection" markedly increased (twice) lipofection efficiency in C6 cells. Expression of beta-GAL reached a maximum after 3-5 days. When the liposome-DNA complexes were injected/infused directly into the brains of adult rats, several weakly stained cells could be observed in the brain region adjacent to the injection site. It is concluded that liposome-mediated gene transfer is an efficient method for gene transfer into CNS cells in vitro, but the transfection efficiency into the rat brain in vivo is far too low and therefore not applicable.