Beta-galactosidase Marker Gene Research Articles

TDP-43 Is a Developmentally Regulated Protein Essential for Early Embryonic Development

TDP-43 is a DNA/RNA-binding protein implicated in multiple steps of transcriptional and post-transcriptional regulation of gene expression. Alteration of this multifunctional protein is associated with a number of neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin positive inclusions. Whereas a pathological link to neurodegenerative disorders has been established, the cellular and physiological functions of TDP-43 remain unknown. In this study, we show that TDP-43 is a nuclear protein with persistent high-level expression during embryonic development and with progressively decreased protein levels during postnatal development. In mice where the TDP-43 gene (Tardbp) was disrupted using a gene trap that carries a beta-galactosidase marker gene, heterozygous (Tardbp(+/-)) mice are fertile and healthy, but intercrosses of Tardbp(+/-) mice yielded no viable homozygotic null (Tardbp(-/-)) mice. Indeed, Tardbp(-/-) embryos die between 3.5 and 8.5 days of development. Tardbp(-/-) blastocysts grown in cell culture display abnormal expansion of their inner cell mass. The pattern of beta-galactosidase staining at E9.5 Tardbp(+/-) embryos is predominantly restricted to the neuroepithelium and remains prominent in neural progenitors at E10.5-12.5. TDP-43 is detected in spinal cord progenitors and in differentiated motor neurons as well as in the dorsal root ganglia at E12.5. Beta-galactosidase staining of tissues from adult Tardbp(+/-) mice shows widespread expression of TDP-43, including prominent levels in various regions of the central nervous system afflicted in neurodegenerative disorders. These results indicate that TDP-43 is developmentally regulated and indispensible for early embryonic development.

Potent Inhibition and Global Co-localization Implicate the Transmembrane Kunitz-type Serine Protease Inhibitor Hepatocyte Growth Factor Activator Inhibitor-2 in the Regulation of Epithelial Matriptase Activity

Hepatocyte growth factor activator inhibitors (HAI)-1 and -2 are recently identified and closely related Kunitz-type transmembrane serine protease inhibitors. Whereas HAI-1 is well established as an inhibitor of the serine proteases matriptase and hepatocyte growth factor activator, the physiological targets of HAI-2 are unknown. Here we show that HAI-2 displays potent inhibitory activity toward matriptase, forms SDS-stable complexes with the serine protease, and blocks matriptase-dependent activation of its candidate physiological substrates proprostasin and cell surface-bound pro-urokinase plasminogen activator. To further explore the potential functional relationship between HAI-2 and matriptase, we generated a transgenic mouse strain with a promoterless beta-galactosidase marker gene inserted into the endogenous locus encoding HAI-2 protein and performed a global high resolution mapping of the expression of HAI-2, matriptase, and HAI-1 proteins in all adult tissues. This analysis showed striking co-localization of HAI-2 with matriptase and HAI-1 in epithelial cells of all major organ systems, thus strongly supporting a role of HAI-2 as a physiological regulator of matriptase activity, possibly acting in a redundant or partially redundant manner with HAI-1. Unlike HAI-1 and matriptase, however, HAI-2 expression was also detected in non-epithelial cells of brain and lymph nodes, suggesting that HAI-2 may also be involved in inhibition of serine proteases other than matriptase.

Induction of Tachykinin Production in Airway Epithelia in Response to Viral Infection

BackgroundThe tachykinins are implicated in neurogenic inflammation and the neuropeptide substance P in particular has been shown to be a proinflammatory mediator. A role for the tachykinins in host response to lung challenge has been previously demonstrated but has been focused predominantly on the release of the tachykinins from nerves innervating the lung. We have previously demonstrated the most dramatic phenotype described for the substance P encoding gene preprotachykinin-A (PPT-A) to date in controlling the host immune response to the murine gammaherpesvirus 68, in the lung.Methodology/Principal FindingsIn this study we have utilised transgenic mice engineered to co-ordinately express the beta-galactosidase marker gene along with PPT-A to facilitate the tracking of PPT-A expression. Using a combination of these mice and conventional immunohistology we now demonstrate that PPT-A gene expression and substance P peptide are induced in cells of the respiratory tract including tracheal, bronchiolar and alveolar epithelial cells and macrophages after viral infection. This induction was observed 24h post infection, prior to observable inflammation and the expression of pro-inflammatory chemokines in this model. Induced expression of the PPT-A gene and peptide persisted in the lower respiratory tract through day 7 post infection.Conclusions/SignificanceNon-neuronal PPT-A expression early after infection may have important clinical implications for the progression or management of lung disease or infection aside from the well characterised later involvement of the tachykinins during the inflammatory response.

Efficient transfer of embryonic stem cells into the cochlea via a non-invasive vestibular route

Conclusion. Cell transplantation into the utriculus provides an efficient and non-invasive route to introduce embryonic stem (ES) cells into the vestibular and cochlear portions of the inner ear. Objective. The transfer of stem cells into the inner ear for therapeutic purposes is an important approach to cure damage to the cochlea and vestibulum. A key issue is to provide an entry point for cell transplants into the inner ear that does not affect its physiologic functions. The aim of this study was to examine the feasibility of transferring ES cells into the inner ear via the utriculus. Materials and methods. ES cells were injected via utriculostomy into the mouse inner ear. The distribution of the injected cells was determined using a beta-galactosidase marker gene expressed by the ES cells. Results. Injected ES cells were found within the perilymph of the scala tympani and vestibuli. Moreover, ES cells were detected close to the cochlear sensory epithelium and spiral limbus.

Optimization of adenoviral vector-mediated transgene expression in the canine brain in vivo, and in canine glioma cells in vitro

Expression of the immune-stimulatory molecule Fms-like tyrosine kinase 3 ligand (Flt3L) and the conditional cytotoxic enzyme herpes simplex virus type 1 thymidine kinase (HSV1-TK) provides long-term immune-mediated survival of large glioblastoma multiforme (GBM) models in rodents. A limitation for predictive testing of novel antiglioma therapies has been the lack of a glioma model in a large animal. Dogs bearing spontaneous GBM may constitute an attractive large-animal model for GBM, which so far has remained underappreciated. In preparation for a clinical trial in dogs bearing spontaneous GBMs, we tested and optimized adenovirus-mediated transgene expression with negligible toxicity in the dog brain in vivo and in canine J3T glioma cells. Expression of the marker gene beta-galactosidase (beta-Gal) was higher when driven by the murine (m) than the human (h) cytomegalovirus (CMV) promoter in the dog brain in vivo, without enhanced inflammation. In the canine brain, beta-Gal was expressed mostly in astrocytes. beta-Gal activity in J3T cells was also higher with the mCMV than the hCMV promoter driving tetracycline-dependent (TetON) transgene expression within high-capacity adenovirus vectors (HC-Ads). Dog glioma cells were efficiently transduced by HC-Ads expressing mCMV-driven HSV1-TK, which induced 90% reduction in cell viability in the presence of ganciclovir. J3T cells were also effectively transduced with HC-Ads expressing Flt3L under the control of the regulatable TetON promoter system, and as predicted, Flt3L release was stringently inducer dependent. HC-Ads encoding therapeutic transgenes under the control of regulatory sequences driven by the mCMV promoter are excellent vectors for the treatment of spontaneous GBM in dogs, which constitute an ideal preclinical animal model.

Co‐localization of the channel activating protease prostasin/(CAP1/PRSS8) with its candidate activator, matriptase

Prostasin (CAP1/PRSS8) is a glycosylphosphatidylinositol-anchored membrane serine protease believed to be critical for the regulation of epithelial sodium channel (ENaC) activity. Prostasin is synthesized as an inactive zymogen that requires a site-specific endoproteolytic cleavage to be converted to an active protease. We have recently reported that the tumor-associated type II transmembrane serine protease, matriptase is necessary and sufficient for prostasin activation in the epidermis. In this study, the interrelationship between the two membrane serine proteases was investigated further by using enzymatic gene trapping combined with immunohistochemistry to delineate the spatial expression of matriptase and prostasin in mouse tissues. We utilized a knock-in mouse with a promoterless beta-galactosidase marker gene inserted into the matriptase locus, as a unique tool for precise assessment of endogenous matriptase expression. The spatial expression of matriptase and prostasin in mouse tissues was delineated by combining in situ beta-galactosidase matriptase staining with immunohistochemical detection of prostasin. We report that prostasin displays a near-ubiquitous co-localization with its candidate activator matriptase in a variety of normal epithelial tissues. These include simple, stratified, and pseudo-stratified epithelium of the integumentary system, digestive tract, respiratory tract, and urogenital tract. However, matriptase and prostasin expression segregates during epithelial multi-stage carcinogenesis to eventually become localized in separate compartments of the tumor. These data suggest that a matriptase-prostasin zymogen activation cascade may be functionally operative in multiple epithelial tissues, but matriptase promotes epithelial carcinogenesis independent of prostasin.

Adenovirus gene transfer of recombinant endothelial nitric oxide synthase enhances contractile function in ventricular myocytes.

eNOS is expressed in cardiac myocytes and plays an important role in cardiac contractile function. This study was designed to determine whether ex vivo eNOS gene transfer in ventricular myocytes affects cardiac contractile function. Replication-incompetent adenoviral vectors encoding eNOS or marker gene beta-galactosidase (LacZ) were transduced into adult rat ventricular myocytes at an MOI of 10, 50, or 100 for 36 hours. Mechanical and intracellular Ca2+ properties of myocytes were evaluated by video-based edge detection and fura-2 fluorescence. NOS protein expression and activity were assessed by Western blot and 3H-arginine to 3H-citrulline assay. Myocytes transduced with eNOS but not LacZ displayed enhanced eNOS but not iNOS expression associated with elevated NOS activity. Myocytes transduced with eNOS exhibited significantly elevated peak shortening and velocity of shortening/relengthening associated with enhanced basal as well as electrically stimulated rise of intracellular Ca2+ compared with control or LacZ groups. The durations of shortening and relengthening were comparable in all groups. The eNOS-induced mechanical effects were paralleled with elevated phosphorylation of Akt. Furthermore, the phosphatidylinositol-3 (PI-3) kinase inhibitors wortmannin and LY294002 prevented eNOS-induced mechanical effects. These results revealed that gene transfer of eNOS directly promotes cardiomyocyte contractile function and intracellular Ca2+ handling, suggesting therapeutic potential of eNOS gene transfer.

Enhancement of spine fusion using combined gene therapy and tissue engineering BMP-7-expressing bone marrow cells and allograft bone.

Prospective study to assess the enhancement of spine fusion using a tissue engineering construct consisting of bone marrow cells genetically modified by adenovirus (Ad) vector-encoding bone morphogenetic protein-7 (BMP-7) seeded onto an allograft scaffold in a rat model. To evaluate Ad transgene expression at the fusion site and the effect of AdBMP-7-treatment on fusion rates, mechanical stability, microscopic anatomy, and bone formation rates. Nonunion is a major complication of spine fusion. Gene transfer may be an effective method for locally overexpressing BMP-7, a gene important for bone formation and regeneration to enhance allograft spine fusion. Bone marrow cells were treated with AdBMP-7 or Adbetagal (encoding the marker gene beta-galactosidase), AdNull (with no gene), or no vector and implanted with allograft in a site of posterior spine fusion. Marker gene expression was assessed up to 14 days after administration. Fusions were evaluated at 8 weeks. Ad gene expression was maximal on day 3, waning to background levels by 14 days. With AdBMP-7 treatment, radiographic fusion rate was 70% and mechanical fusion rate was 80% versus 0% by either parameter in control groups. Fused AdBMP-7-treated spines had a 2.5-fold to 3.0-fold lower range of motion and 1.7-fold to 1.9-fold lower hysteresis than controls. Fusion masses of AdBMP-7-treated spines had the microscopic appearance of normal trabecular bone and showed a 23-fold higher uptake of fluorochrome indicating increased bone formation. Addition of AdBMP-7-modified marrow cells can enhance allograft spine fusion.

Infection Efficiency of Human and Mouse Embryonic Stem Cells Using Adenoviral and Adeno-Associated Viral Vectors

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.

Muscle cell-mediated gene delivery to the rotator cuff.

Rotator cuff lesions are one of the most common causes of upper extremity disability. Surgical therapy addresses mostly the extrinsic etiology, but not intrinsic factors such as aging, structural changes, low vascularity, and inflammatory processes. In this study, genetically engineered, highly purified muscle-derived cells (MDCs) were characterized and injected into the supraspinatus tendons of nude rats. The injected cells were monitored for 3 weeks. In vitro, the engineered, highly purified MDCs do not express vimentin; 98% of them are positive for the beta-galactosidase marker gene, and 99% hybridize with the specific pancentromeric mouse probe. beta-Galactosidase marker gene expression of the injected cells was detected up to 21 days. From day 7 after injection, the cell nuclei became spindle shaped, cells were integrated into the tendon collagen bundles, and the cells showed differentiation into vimentin-expressing fibroblastic cells. The results indicate that the rotator cuff tendon matrix and its original cellular components modulated the injected MDCs toward a fibroblastic phenotype. The compatibility and ability of MDCs to differentiate into other cell lineages, such as fibroblasts, might have high potential utility in tissue-engineering applications for tendon healing. This approach facilitates the application of muscle-derived progenitor cells and ex vivo gene therapy for the treatment of rotator cuff lesions.

Adeno-associated virus-mediated osteoprotegerin gene transfer protects against particulate polyethylene-induced osteolysis in a murine model.

Osteoprotegerin (OPG), a natural negative regulator of osteoclastogenesis and bone resorption, may be a potential therapeutic agent for treatment of osteolysis-associated prosthetic joint loosening. Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study was designed to evaluate the protective effects of OPG transgene against orthopedic wear debris-induced bone loss in a murine model of osteolysis. Bone tissue was implanted into established pouches on BALB/c mice, followed by the introduction of ultra-high-molecular-weight polyethylene (UHMWPE) particles to provoke inflammation and osteolysis. The viruses encoding human OPG gene (rAAV-hOPG) or beta-galactosidase marker gene (rAAV-LacZ) were injected into the air pouches, and the tissue was harvested 7 days after viral infection for histologic and molecular analyses. Successful transgene expression was confirmed by the detection of OPG by enzyme-linked immunosorbent assay and positive X-Gal staining of pouch tissue (LacZ). Real-time polymerase chain reaction indicated significant diminishment of messenger RNA expression of osteoclast markers in OPG-transduced pouches compared with rAAV-LacZ-transduced pouches. The transduction and expression of OPG also markedly decreased the gene copies of the biologic receptor activator of nuclear factor kappaB. The expression of OPG in the bone-implanted pouch reduced bone calcium release by a mean of 39% compared with the calcium release in the other 2 groups. Computerized image analysis revealed that expression of OPG significantly protected against bone collagen loss. OPG gene transfer mediated by rAAV effectively protects against particulate polyethylene-induced bone resorption in this experimental model. Data suggest that gene transfer using rAAV-OPG may be a feasible and effective therapeutic candidate to treat or prevent wear debris-associated osteolysis and aseptic loosening.

Adenovirus-mediated gene transfer in the ovine pituitary gland is associated with hypophysitis.

Gene therapy for pituitary disease requires evaluation for safety as well as efficacy. We have reported results of adenovirus-mediated gene transfer using the sheep as a large animal model that allows longitudinal evaluation of hormone secretion and have confirmed high levels of transgene expression up to 7 days after direct stereotaxic injection into the pituitary gland. Here we report the results of detailed histological examination of the pituitary glands from animals injected with two recombinant adenoviruses expressing the beta-galactosidase marker gene, or with saline vehicle to control for the potential tissue-disruptive effect of the injection volume itself. Pituitaries injected with saline showed no evidence of inflammatory response apart from occasional minor foci of apoptosis. In all other respects they were indistinguishable from normal uninjected control pituitary glands. Glands injected with recombinant adenoviruses containing either the hCMV-beta-gal or the hPRL-beta-gal transgene, on the other hand, displayed variable degrees of inflammatory response, with periglandular fibrosis, lymphocytic infiltrate and venulitis in almost all cases. Focal necrosis and/or apoptosis was noted in six of nine cases. In summary, we have found evidence of severe inflammatory reaction within the first seven days of adenovirus injection, amounting to significant hypophysitis. The histological extent of this reaction has not previously been recognised by studies of the efficacy of gene transfer in rodents, and was underestimated by immunocytochemical studies of hormone and transgene expression. The findings emphasise the need for careful evaluation of the safety of endocrine gene therapy, and for caution with the dose of vector used.

18] Gene therapy methods in cardiovascular diseases

Local gene transfer into the vascular wall offers a promising alternative to treat atherosclerosis-related diseases. Blood vessels are among the easiest targets for gene therapy because of percutaneous, catheter-based treatment methods. On the other hand, gene transfer to the artery wall can also be accomplished from adventitia either by ex vivo gene transfer and implantation of transfected cells or by direct in vivo gene transfer methods. In the future, as the pathological processes in arteries are better understood, several therapeutic genes could be combined and these "gene cocktails" are expected to produce enhanced therapeutic effects in vascular gene therapy. We have developed a new, efficient technique for performing ex vivo gene transfer to rabbit arterial wall using autologous SMC. The cells were harvested from rabbit ear artery, transfected in vitro with VSV-G pseudotyped lacZ retrovirus, and returned back to the adventitial surface of the carotid artery using a silicone collar or collagen sheet placed around the artery. The transduced SMCs implanted with a high efficiency and expressed beta-galactosidase marker gene at a very high level 7 days and 14 days after the operation. The level of lacZ expression decreased thereafter, but was still easily detectable for at least 6 months and was exclusively localized to the site of cell implantation inside the collar. Development of new vectors, such as baculovirus, for gene transfer will provide targeted, efficient, and safer methods for gene delivery. Plasmids and viruses coding for more than one protein, and bearing regulatory elements, would be useful for future gene therapy applications. Also, constructing second-generation viruses that contain fewer endogenous genes in their genome may reduce immunological reactions caused by the first-generation adenoviruses. In conditions where stable expression of therapeutic proteins is needed, it is necessary to develop better ex vivo and in vivo gene transfer strategies. Also, production of viruses that can efficiently transfect nondividing cells will be important for future applications of vascular gene therapy. However, current knowledge from vascular gene transfer experiments strongly suggests that vascular gene transfer is a promising new alternative for the treatment of cardiovascular diseases.

New use of a three-dimensional pellet culture system for human intervertebral disc cells: initial characterization and potential use for tissue engineering.

Human intervertebral disc cells were cultured in a new three-dimensional "pellet culture" system as an alternative to conventional alginate bead microspheres. Histologic, biochemical, and immunohistologic assays were performed to characterize this new culturing method for disc cells. The feasibility of using the pellet culture system to study effects of gene therapy was also assessed. To characterize a new and simpler, three-dimensional culture system for human intervertebral disc cells and to assess the feasibility of its use for gene therapy and tissue engineering studies. The alginate microsphere three-dimensional culture system has been the most utilized culture method for disc cells, but it is technically difficult and has some disadvantages. Recently, the "pellet culture" method, a simpler three-dimensional culture system, was described for bone marrow stromal cells. This simpler method might be useful for in vitro and in vivo studies of disc cells as well as for delivery of exogenous genes. Isolated human intervertebral disc cells were centrifuged at low speeds to form aggregates and allowed to grow as pellets for up to 3 weeks. At various times these pellet cultures were analyzed grossly, histologically, and immunohistologically. Their ability to incorporate [35S]sulfate in their response to TGF-beta1 was also analyzed. The ability of these pellets to deliver and express exogenous genes in vivo was analyzed by implantation of pellet cultures in muscles of SCID mice. Within several days the intervertebral disc cells were able to form mature three-dimensional aggregates that were each well encapsulated by a fibrous capsule. These pellets successfully synthesized proteoglycan and collagen Type II matrix as determined by histology and immunohistochemistry. In response to TGF-beta1, the pellets increased synthesis of proteoglycan and collagen Type II. When implanted into thigh muscles of SCID mice, the pellets remained aggregated and expressed the beta-galactosidase marker gene in vivo for up to 2 weeks. The pellet culture system is a new and technically simple method to culture human intervertebral disc cells. The majority of the human intervertebral disc cells retained their native phenotype in this three dimensional system and expressed a marker gene both in vitro and in vivo. Thus, this pellet system might be useful for in vitro and in vivo biochemical studies as well as for studies involving gene therapy and tissue engineering.

Switching on and off transgene expression within lactotrophic cells in the anterior pituitary gland in vivo.

To further develop our understanding of anterior pituitary (AP) function and to aid the development of gene therapy strategies for the treatment of pituitary diseases, adenovirus (Ad)-mediated gene transfer to the AP gland will be a useful tool. Although successful widespread gene transfer within the AP has been achieved using first generation Ads the ability to control transgene expression would be very beneficial when studying AP regulatory functions and delivering a potentially therapeutic gene into the AP gland. A dual adenoviral vector system encoding for cell type-specific and regulatable transcription units was developed to achieve transcriptionally targeted transgenesis within specific cell populations in the adult AP gland. To achieve regulatable transgene expression within predetermined AP cells, the tetracycline-responsive transcriptional elements have been engineered to be under the control of human, lactotroph-specific PRL (hPRL) promoter elements within a dual adenoviral vector system. The inducibility, cell type specificity, and levels of transgene expression were characterized in vitro and in vivo and compared with the strong ubiquitous beta-actin/human cytomegalovirus (CAG) promoter. Inducible expression of the marker gene beta-galactosidase under the control of the hPRL promoter was restricted to lactotrophic tumor cell lines and lactotrophic cells within primary AP cultures. Lactotroph cell type specificity and inducible transgene expression were also observed within the AP gland in vivo, and this could be switched on or off. Administration of doxycycline abrogated transgene expression both in vitro and in vivo. Our results also provide evidence that an excess of trans-activator is needed to achieve maximal transgene expression. Our data indicate that combined transcriptional and inducible transgenesis can be achieved using adenoviral vectors that allow spatial and temporal restriction of transgene expression within the adult AP gland in vivo.

Baculovirus-mediated periadventitial gene transfer to rabbit carotid artery.

Recombinant Autographa californica multiple nuclear polyhedrosis viruses (AcMNPV) have recently been shown to transduce mammalian cells in vitro. Since baculoviruses offer many advantages over viruses currently used in gene therapy, we have tested them for in vivo gene transfer by constructing a baculovirus bearing a nuclear targeted beta-galactosidase marker gene (LacZ) under a CMV promoter. Both rabbit aortic smooth muscle cells (RAASMC) and human ECV-304 cells were susceptible to LacZ-baculovirus transduction. Transgene expression was evaluated in vivo by applying 1 x 10(9) p.f.u. of LacZ-baculoviruses or LacZ-adenoviruses in a silastic collar placed around rabbit carotid arteries in the absence of contact with blood components. As a result, baculoviruses led to transgene expression in adventitial cells in rabbit carotid arteries with efficiency comparable to adenoviruses. The beta-galactosidase gene expression was transient staying at a high level for 1 week but disappearing at the 14 day time-point. The arterial structure and endothelium remained intact in the baculovirus-transduced arteries, but macrophage-specific immunostaining detected signs of inflammation comparable to adenoviruses. Baculoviruses are thus able to mediate transient gene transfer in vivo and may become useful tools for gene therapy.

In vivo transfer of bacterial marker genes results in differing levels of gene expression and tumor progression in immunocompetent and immunodeficient mice.

To optimize gene delivery for the treatment of malignant mesothelioma, expression of the beta-galactosidase marker gene was examined in a murine model of intraperitoneal malignant mesothelioma. The beta-galactosidase gene was delivered to the peritoneal cavity of tumor-bearing mice by various plasmid-liposome complexes or by replication-incompetent retrovirus, used alone or complexed to liposomes. In tumor samples from immunodeficient nude mice, moderate levels of gene expression were achieved by liposome-complexed plasmids. Retroviral gene delivery was more effective, and was increased nearly 10-fold by complexing the retrovirus to liposomes. In contrast, in tumor samples from immunocompetent CBA mice treated with the same vectors, no marker gene expression was detected. In immunodeficient mice, tumor growth was not affected by beta-galactosidase gene transfer. However, immunocompetent mice showed a significant decrease in tumor size and increase in survival time after beta-galactosidase delivery. Induction of cytotoxic T cells capable of lysing beta-Gal-transfected tumor cells suggests that tumor cells transduced with the bacterial beta-galactosidase gene may be eliminated in immunocompetent hosts. Our findings also indicate that plasmid-liposome complexes, which achieve a low level of gene expression, and retrovirus-liposome complexes, which result in nearly 100 times higher levels of gene expression in tumor cells in vivo, are similarly effective in inducing an antitumor immune response.

Adenovirus-mediated gene transfer to lower limb artery of patients with chronic critical leg ischemia.

Arterial gene transfer offers a promising new approach for the treatment of vascular disorders. However, no data are available about the gene transfer efficiency in human arteries in vivo. The aim of this study was to evaluate the safety and feasibility of catheter-mediated adenoviral gene transfer in human peripheral arteries. Ten patients (8 females, 2 males, mean age 80 +/- 8 years) suffering from chronic critical leg ischemia with a prior decision for amputation were recruited in the study. Gene transfer was performed in eight patients in conjunction with a conventional percutaneous transluminal angioplasty, using a perfusion coil balloon catheter. Two patients served as controls. Increasing concentrations of replication-deficient adenoviruses (titers from 1 x 10(8) to 4 x 10(10) PFU) containing a nuclear-targeted beta-galactosidase marker gene were administered into the arteries over 10 min via the catheter. Amputations were performed 20 to 51 hr after the procedures and gene transfer efficiency was evaluated in the transduced arteries using X-Gal staining for beta-galactosidase activity. Beta-galactosidase gene transfer was well tolerated and no adverse tissue responses or systemic complications were observed in any of the patients. Gene transfer was successful in six of the eight patients. Gene transfer efficiency varied between 0.04 and 5.0% of all arterial cells. Transgene expression was detected in smooth muscle cells, endothelial cells, and macrophages and in tunica adventitia. However, transgene activity was not evenly distributed in the arterial wall and no transgene activity was found beneath advanced atherosclerotic lesions. The safety and feasibility of in vivo gene transfer by adenoviral vectors to human peripheral arteries were established. Although improvements are still required in gene transfer efficiency, these findings suggest that adenoviruses can be used to deliver therapeutically active genes into human arteries.

Efficiency assessment of the gene trap approach.

The trapping of genes in murine embryonic stem (ES) cells offers three features in one experimental approach: 1) analysis of the expression patterns of unknown genes by using a simple staining method, 2) rapid cloning of unknown genes, and 3) generation of mutant mouse lines. We performed a gene trap screen aimed at the discovery of new genes regulating embryonic development. We have processed 209 gene trap events for expression patterns in chimeric murine embryos. Randomly tested, beta-galactosidase-positive ES cell clones resulted in vivo in 35% gene trap events showing no beta-galactosidase activity, 39% gene trap events with ubiquitous beta-galactosidase activity, and 26% gene trap events showing beta-galactosidase activity restricted to specific cell types or organs. In vitro preselection reduced gene trap events with ubiquitous beta-galactosidase activity to 10% and increased the gene trap events with restricted beta-galactosidase activity to 64%, making the screening procedure for genes expressed in a restricted manner 2.5-fold more efficient. In five of the seven gene trap insertions into genes in which the expression pattern during embryogenesis was known, the beta-galactosidase marker gene reproduced faithfully the expression pattern of the trapped gene. 5'-Rapid amplification of cDNA ends (5'-RACE) of 28 gene trap events revealed 19 novel mouse genes, 8 known mouse genes, and 1 random transsplicing event. Twelve of the 25 mouse lines that crossed to homozygosity showed overt abnormalities. The genomic structure was investigated in four of these gene trap events, which caused obvious abnormalities. In all four cases, the splice-acceptor gene trap construct was inserted into an exon. One of the 13 gene trap events that did not result in overt abnormalities was examined for the presence of wild-type mRNA. Homozygous animals were found to produce normal levels of wild-type mRNA. Evidently, gene trapping does not always provide all three of the features mentioned above. In this paper, we discuss the efficiency of gene trapping and ways in which some problems may be overcome.

Bodenin: a novel murine gene expressed in restricted areas of the brain.

A gene trap screen designed to isolate novel mouse genes involved in nervous system development was performed. Here, we describe the isolation and characterization of a novel gene, bodenin, which is expressed in restricted areas of the brain. beta-Galactosidase marker gene activity was detected in the embryo at the start of organogenesis (embryonic day 8.5; E8.5). Staining gradually became stronger until E12.5, when embryos exhibited widespread expression. In brains of newborn and adult mice, beta-galactosidase staining was confined predominantly to forebrain structures. Transcriptional activity was also observed in kidney, liver, lung, heart, skeletal muscle, and testes. Part of the trapped gene was isolated by 5'-rapid amplification of cDNA ends (5'-RACE). Isolation and sequencing of the complete cDNA revealed an unknown gene encoding a 200 amino acid protein. Comparison with published sequences showed 94% amino acid identity to a human integrin cytoplasmic domain-associated protein. Mice homozygous for the mutation were viable and did not exhibit any obvious abnormal phenotype. However, concealed phenotypic abnormalities cannot be excluded. The lack of readily visible abnormalities may also be due to functional compensation or to the production of low levels of wild-type protein in mice homozygous for the gene trap vector insertion. Nevertheless, the restricted expression of bodenin in the brain of newborn and adult mice suggests a role for this novel gene in the developing and mature central nervous system.