Nonviral gene transfer for studying signaling in comparative developmental biology.
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Biomedical subjects
B A Demeneix
Publications and source records attributed to B A Demeneix.
Studying amphibian metamorphosis by in vivo gene transfer.
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T3 treatment increases mitosis, then bax expression and apoptosis in the optic lobe of the chick embryo.
We investigated T3 effects on cell proliferation and apoptosis in the optic lobe of the chick embryo between embryonic days (E) 6 and 11. Injection of T3 into the yolk increased [3H]thymidine incorporation between E7 and E9. This increased mitosis was followed by altered timing and degree of apoptosis during E9-11. In T3-treated embryos a marked increase in apoptosis occurred on E10, coincident with increased levels of mRNA encoding Bax, a pro-apoptotic protein. By E11, the overall morphology and total number of cells in each layer of the optic lobe were not different in control and treated embryos. Thus, although T3 transiently increases cell number, a homeostatic mechanism enters into play re-adjusting the balance between cellular proliferation and cell death.
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Tail regression, apoptosis and thyroid hormone regulation of myosin heavy chain isoforms in Xenopus tadpoles.
T3 effects on myosin heavy chain gene expression were analysed in muscles undergoing different fates during metamorphosis. Muscle fate was followed by somatic gene transfer of a constitutively expressed luciferase vector. Persistent expression was found in dorsal muscle which is remodelled during metamorphosis whilst the signal disappeared in apoptosing caudal muscle. RNAse protection assay was used to follow production of myosin heavy chain isoforms: two isoforms identified as embryonic (E3 and E19) and one adult form (A7). The effects of T3 treatment were followed over 120 h. During this time frame E3 and A7 expression patterns were found to be similar in both caudal and dorsal muscles. Most notably, at 48 h E3 expression was significantly down-regulated and production of A7 significantly upregulated in both caudal and dorsal muscle. Thus T3-induced transitions in muscle gene expression are independent of muscle fate during amphibian metamorphosis.
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Adenovirus enhancement of polyethylenimine-mediated transfer of regulated genes in differentiated cells.
Efficient gene transfer is a prerequisite for analysing regulation of transfected promoters. We combined the DNA binding property of the cationic polymer polyethylenimine (PEI) and the potent endocytic activity of adenovirus in a PEI-DNA-adenovirus complex which provided efficient plasmid delivery in differentiated cultured cells. We transfected 3T3-F442A adipocytes, C2.7 myocytes and FAO hepatoma cells with a construct containing the simian virus 40 promoter fused to the chloramphenicol acetyltransferase (CAT) gene, using a combination of PEI and 200 p.f.u. per cell of replication-deficient type 5 adenovirus. Resulting CAT activities varied according to the cell type reaching about 0.6, 8 and 38 units/mg protein for respectively 3T3-F442A, FAO and C2.7 cells. Increases in transfection efficiencies were 140- to 300-fold when compared with those obtained with PEI alone. Then we tested physiologically regulated promoters: the phosphoenolpyruvate carboxykinase gene promoter in 3T3-F442A or FAO cells and the hexokinase II gene promoter in C2.7 myocytes. Gene expression was appropriately increased by clofibrate, dexamethasone and insulin for 3T3-F442A, FAO and C2.7 cells, respectively. Thus, the combination of PEI and adenovirus is a simple, efficient, inexpensive and versatile method of gene transfer which is applicable to several differentiated cells and provides a physiologically coherent transgene regulation. We name this method PEI-adenofection.
Adenoviridae↗
Apoptosis in Xenopus tadpole tail muscles involves Bax-dependent pathways.
Apoptosis is a fundamental mechanism implicated in normal development. One of the most spectacular developmental events involving apoptosis is tail regression during amphibian metamorphosis. We analyzed how thyroid hormone (3, 5, 3'-triiodothyronine, T3), the orchestrator of metamorphosis, affects expression and function of the proapoptotic gene Bax in the tail muscle of free-living Xenopus tadpoles. During natural metamorphosis Bax mRNA was expressed in tail muscles and was spatially correlated with apoptosis. Precocious treatment of tadpoles with T3 induced Bax expression and apoptosis. To verify that Bax expression was causally related to apoptosis we used a naked DNA gene transfer method to express Bax in the dorsal tail muscle. This induced apoptosis, and the process was exacerbated by T3 treatment. To determine whether T3 effects on Bax expression involved transcriptional regulation, we injected a Bax promoter sequence into dorsal and caudal tail muscles. In the dorsal muscle, T3 treatment did not affect transcription from the Bax promoter. However, in the caudal muscle, T3 treatment significantly increased Bax transcription. We conclude that T3-induced apoptosis in Xenopus tadpole tail muscles involves Bax-activating and Bax-synergis tic mechanisms. These programs are induced in spatially and temporally distinct manners.
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A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine.
Nonviral gene transfer into the central nervous system (CNS) offers the prospect of providing safe therapies for neurological disorders and manipulating gene expression for studying neuronal function. However, results reported so far have been disappointing. We show that the cationic polymer polyethylenimine (PEI) provides unprecedentedly high levels of transgene expression in the mature mouse brain. Three different preparations of PEI (25-, 50-, and 800-kD) were compared for their transfection efficiencies in the brains of adult mice. The highest levels of transfection were obtained with the 25-kD polymer. With this preparation, DNA/PEI complexes bearing mean ionic charge ratios closest to neutrality gave the best results. Under such conditions, and using a cytomegalovirus (CMV)-luciferase construction, we obtained up to 0.4 10(6) RLU/microgram DNA (equivalent to 0.4 ng of luciferase), which is close to the values obtained using PEI to transfect neuronal cultures and the more easily transfected newborn mouse brain (10(6) RLU/microgram DNA). Widespread expression (over 6 mm3) of marker (luciferase) or functional genes (bcl2) was obtained in neurons and glia after injection into the cerebral cortex, hippocampus, and hypothalamus. Transgene expression was found more than 3 months post-injection in cortical neurons. No morbidity was observed with any of the preparations used. Thus, PEI, a low-toxicity vector, appears to have potential for fundamental research and genetic therapy of the brain.
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GABAB receptors negatively regulate transcription in cerebellar granular neurons through cyclic AMP responsive element binding protein-dependent mechanisms.
GABAB receptors affect short-term signalling in various cell types. However, nothing is known about possible long-term effects on transcription. To analyse such effects in the CNS, we studied GABAB receptor-mediated gene regulation in primary cultures of cerebellar granule neurons. Transcription was followed using a chloramphenicol acetyl transferase reporter gene driven by the minimal cyclic AMP-responsive element (TGACGTCA). Transcription was stimulated by activation of both the cyclic AMP (forskolin: 5 x 10(-6) M) and the Ca2+ dependent (KCl: 30 mM) pathways (-)-Baclofen (10(-6) M to 10(-4) M), a specific GABAB receptor agonist, reduced by 50-70% the transcriptional stimulation evoked by both forskolin and KCl, whereas isoguvacine, a GABAA receptor agonist, was without effect. Moreover, the GABAB antagonist CGP 35348 abrogated the inhibitory effects of both GABA and baclofen, indicating that GABAB receptors were specifically implicated in this response. Measurements of cyclic AMP levels suggested that (-) baclofen inhibits forskolin-initiated transcription by reducing cyclic AMP production. Direct transcriptional activation, via the cyclic AMP pathway, by overexpression of the catalytic subunit of the cyclic AMP-dependent protein kinase, was not significantly altered by (-) baclofen. This indicates again that (-) baclofen-dependent inhibitory mechanisms operate upstream of cyclic AMP-dependent protein kinase at the level of second messenger formation. Further, we used a yeast transcriptional activator GAL4-cyclic AMP-responsive element binding protein to analyse whether GABAB receptor-mediated inhibition of cyclic AMP-responsive element transcription implicated the transacting factor cyclic AMP-responsive element binding protein. We show that the negative effects of (-) baclofen implicate this transcription factor and this holds good for both the forskolin and KCl-stimulated pathways. The results indicate that GABAB receptors negatively regulate cyclic AMP-responsive element binding protein-mediated transcription in the CNS.
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Use of heterologous DNA-based gene transfer to follow physiological, T3-dependent regulation of myosin heavy chain genes in Xenopus tadpoles.
In vivo gene transfer and RNase protection assay were used to follow thyroid hormone (T3)-dependent regulation of myosin heavy chain (myHC) genes in Xenopus tadpole dorsal muscle. One embryonic and one adult myHC form were measured by each approach. RNase protection assay showed that T3 decreased expression of endogenous embryonic mRNA (E3), but increased adult (A7) transcripts. Gene transfer showed that T3 exerted transcriptional effects on mammalian embryonic and adult myHc promoters injected into the same muscle. The kinetics and profiles of the transcriptional responses were superimposable on endogenous responses. The results strengthen the use of in vivo approaches for determining the roles of transcription factors and cis-regulatory sequences in integrated contexts.
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Gene transfer by naked DNA into adult mouse brain.
Nonviral gene transfer into the central nervous system could provide a basis for therapeutic uses and fundamental research. We show that naked DNA injected intracerebrally into the mouse brain can provide expression of a reporter protein. Expression is dose dependent, being maximal for 150 mu g DNA injected. We observed less than 5 days expression of the luciferase transgene, which is not improved with plasmid preparations virtually free of lipopolysaccharide. Thus, the adult brain behaves as striated muscle for naked DNA uptake and transcription, albeit at a much lower efficiency. In neither adult brain nor muscle did complexation of DNA with cationic lipid improve transgene expression. Double immunolabeling using cell-specific markers shows that both neurons and glia are transfected by naked DNA gene transfer methodology.
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Inhibition of neurogenic precursor proliferation by antisense alpha thyroid hormone receptor oligonucleotides.
Thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) is required for normal brain development in vertebrates. T3 acts through two classes of nuclear receptors (TR alpha and TR beta) that have distinct developmental spatial and temporal distributions suggesting different functions during neuronal development. One possibility is that TR alpha, which is expressed early in embryogenesis, is involved in neuroblast proliferation. To test this hypothesis we used the embryonic chick optic lobe, as we found that T3 stimulates [3H]thymidine incorporation in this tissue both in vivo and in vitro during embryonic days 6-9. We applied oligonucleotides (ODNs) against TR alpha and TR beta to primary cultures of chick optic lobes. By employing a cationic lipid vector we could use very low ODN concentrations (< 150 nM). Antisense ODNs against TR alpha significantly inhibited [3H]thymidine incorporation, whereas antisense TR beta had no significant effect. However, both ODNs inhibited expression of TRs, as they blocked transcription from a T3-activated reporter gene. Random ODNs used as controls had no significant effect on [3H]thymidine incorporation or on T3-dependent transcription. These observations suggest that TR alpha is implicated in neuroblast proliferation and add credence to the hypothesis that the multiplicity of nuclear receptors allows for specific actions of T3 during development.
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Lipospermine-based gene transfer into the newborn mouse brain is optimized by a low lipospermine/DNA charge ratio.
Nonviral, plasmid-based gene transfer into somatic tissues offers the prospect of various simple and safe therapeutic possibilities as well as applications in fundamental research. Although cationic lipids display efficient transfection activities in many in vitro systems, only low success rates using these vectors in vivo have been reported. We succeeded in defining conditions providing high levels of in vivo transfection in the brains of newborn mice. Our hypothesis was that conditions favorable for in vitro transfection (highly positively charged particles) were unlikely to be appropriate for in vivo conditions. When using the cationic lipid dioctadecylamido glycylspermine (Transfectam, DOGS) with a cytomegalovirus (CMV)-luciferase reporter gene, the best levels of transfection were obtained when using a low ratio of positive charges (supplied by the DOGS) to negative charges (carried by the DNA). Moreover, addition of the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) significantly enhanced transfection. Expression of the transgene diminished over time, independently of lipopolysaccharide content of the plasmid preparation used. This suggests that either a mitotic population of cells was preferentially transfected, or that promoter silencing was occurring. Histological examination of the spatial distribution of a beta-galactosidase-expressing transgene showed numerous groups of transfected cells both within the striatal parenchyma and in the paraventricular area. Thus, DNA-lipid complexes bearing overall charges close to neutrality open promising possibilities for modulating gene expression in the developing central nervous system and for therapy in the brain.
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Non-viral gene transfer: applications in developmental biology and gene therapy.
The main limitation of non-viral gene transfer methods is their relatively low efficiency in vivo. However, a number of approaches can be taken to improve their performances, whether the aim is studying gene function during development or employing these techniques for gene therapy. Three non-viral delivery systems that we have been particularly involved in in developing are described: the cationic lipid, dioctadecylamidoglycylspermine (DOGS), the cationic polymer polyethylenimine (PEI) and free DNA. The application of each of these methods to different in vivo situations is presented: the use of DOGS for transfecting embryos and the developing mammalian nervous system; the recent application of PEI to the nervous system; and how naked DNA can be employed for transfecting different muscles and brain. The relative efficiencies are compared on the basis of luciferase reporter gene expression assessed in each tissue with the most appropriate vector system. Finally, the perspectives for constructing composite vectors combining safety and efficiency are considered briefly.
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Precocious auto-induction of thyroid hormone receptors in embryonic chick hypothalamic neurons.
Qualitative immunocytochemical and quantitative autoradiographic approaches were used to examine the presence of thyroid hormone receptor (TR) proteins in embryonic chick hypothalamic cultures and the effect of T3 on their expression. We used antibodies raised against rat TRs having first verified that they recognised nuclear T3 binding proteins in extracts from chick brains. TRs were expressed in embryonic hypothalamic neurons from 6 day-old chick embryos maintained 4 days in vitro, and their expression was up-regulated by T3. This demonstration of TRs in embryonic hypothalamic neurons suggests that a physiological mechanism for early regulation of the hypothalamic neuropeptide, thyrotropin releasing hormone, is installed prior to the onset of thyroid function.
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Temporal and spatial expression of lipospermine-compacted genes transferred into chick embryos in vivo.
We have optimized a lipospermine-based transfection method for introducing genes into intact vertebrate embryos in vivo. The method employs small amounts of the cationic lipid Transfectam (DOGS), in a concentrated (40 mM) ethanolic solution, to compact and to transfer exogenous genes into chick embryos during the early stages of development (< 36 h of incubation). Plasmid vectors containing the reporter gene luciferase were used to follow the time course of expression. Luciferase activity was detected as early as 12 h post-transfection and was highest at this time. Enzyme activity then decreased over the next two days and was usually undetectable by 72-h post-transfection. To follow the spatial expression of the exogenous genes, a Rous sarcoma virus (RSV)-beta-galactosidase vector was used. When the transfection complex was applied externally around the developing embryo, the main site of expression was the cardiac tissue. Expression could be targeted to the nervous system by micro-injecting the DNA/DOGS (DNA/dioctadecylamidoglycylspermine) complex into the developing brain. The results show that reporter genes can be efficiently expressed in both the developing central nervous system and heart. This raises the possibility that lipospermines can be used to transfer functional genes into embryos during defined periods of development and also to deliver genes in other species and in other in vivo contexts.
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Excess iodide and thyroid function in the developing chick embryo.
The effects of adding excess iodide (I-) to egg yolks were studied on the quantity of iodide transferred to the chick embryo and on thyroid function from Days 8 to 18 of incubation. Following injection of either 0.5 or 1 mg of iodide into the yolk on the second day of incubation, embryonic plasma iodine levels increased significantly over levels of controls at the same age. These increases were proportional to the amount of excess iodide in the yolk. Moreover, a constant ratio between the iodine levels of the allantoic fluid and the plasma was maintained for any given day of incubation. Up to Day 10, the iodine content of the embryonic thyroid was closely correlated with the increased plasma iodine concentrations. After Day 10, concomitant with the establishment of the hypothalamo-adenohypophyseal-thyroid axis, the increase in thyroid iodine no longer paralleled that of plasma iodine, but was only twice that of controls. As thyroid weight doubled in these treated embryos, thyroid iodine concentrations of control and treated embryos were similar. Iodide excess did not block thyroid iodine organification, nor was there any modification of iodine distribution among the different iodo-amino acids. Moreover, despite the doubling of thyroid hormone content in the goiters of treated embryos, circulating levels of thyroid hormones in control and treated embryos were not different.
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Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression.
Striated muscle is the only tissue found to be capable of taking up and expressing reporter genes that are transferred in the form of plasmid DNA. Thus, direct gene transfer is a potential method of gene therapy for the primary inherited myopathies. However, results to date have had insufficient and too variable expression to consider using direct gene transfer in human trials. We have determined that much of the variability of expression is due to nonuniform distribution of substances injected into skeletal muscle in vivo, and have developed a model to ameliorate this. Preinjection of muscles with a relatively large volume of hypertonic sucrose improves the distribution of injected substances and results in significantly less variable expression of reporter genes for luciferase or beta-galactosidase; the coefficient of variation for mean luciferase activity was reduced from about 120% to 25%. Expression is not directly proportional to dose, but is more so if the muscles are preinjected with sucrose than not. Expression is higher and less variable if DNA is injected in a larger than a smaller volume. The choice of promoter appears to be particularly important. Luciferase reporter gene expression from the SV40 promoter was transient and low, whereas expression driven by the Rous sarcoma virus (RSV) promoter was high and sustained, such that a 1,000-fold difference in expression could be observed. The mechanism of gene uptake is still unknown, but our findings indicate that fibers damaged by the injection procedure do not take up and express plasmid DNA.
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Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle.
Direct gene transfer into skeletal muscle offers several therapeutic possibilities. We assessed direct intramuscular injection of recombinant plasmids, adenovirus, or retrovirus in normal or regenerating muscles of mice. The incorporation and expression of reporter genes introduced by any of these three vectors is greater in regenerating than in mature muscle. In regenerating muscle, pure DNA and adenovirus result in equivalent numbers of fibers expressing reporter gene (> 10%), but adenovirus also induces considerable cellular infiltration. In mature muscle, recombinant DNA is better than adenovirus. Retrovirus failed to infect mature muscle fibers and was less effective than plasmid DNA or adenovirus in regenerating muscle. The surprisingly high relative efficiency of pure plasmid DNA suggests that this method will provide a simple, safe and viable alternative for gene therapy involving muscle tissue.
Adenoviridae↗