Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor.

Interleukins 4 (IL-4) and 13 (IL-13) have been found previously to share receptor components on some cells, as revealed by receptor cross-competition studies. In the present study, the cloning is described of murine NR4, a previously unrecognized receptor identified on the basis of sequence similarity with members of the hemopoietin receptor family. mRNA encoding NR4 was found in a wide range of murine cells and tissues. By using transient expression in COS-7 cells, NR4 was found to encode the IL-13 receptor alpha chain, a low-affinity receptor capable of binding IL-13 but not IL-4 or interleukins 2, -7, -9, or -15. Stable expression of the IL-13 receptor alpha chain (NR4) in CTLL-2 cells resulted in the generation of high-affinity IL-13 receptors capable of transducing a proliferative signal in response to IL-13 and, moreover, led to competitive cross-reactivity in the binding of IL-4 and IL-13. These results suggest that the IL-13 receptor alpha chain (NR4) is the primary binding subunit of the IL-13 receptor and may also be a component of IL-4 receptors.

Identification of an inducible surface molecule specific to fusing macrophages.

Multinucleated giant cells and osteoclasts arise through the fusion of mononuclear phagocyte precursors. To elucidate the mechanism by which cells of monocytic lineage fuse and differentiate into giant cells and osteoclasts, we hypothesized that, as with other cell fusion events, specific surface molecules mediate the adhesion/fusion process. It has been observed that macrophages can be induced to fuse with one another in response to specific stimuli or when placed in a specific microenvironment. The formation of giant cells is primarily associated with chronic inflammatory reactions and tumors, while osteoclasts differentiate on bone which they resorb. The fact that, under normal conditions, macrophages and monocytes fail to fuse in regions and tissues where they are present in large numbers suggests the regulated and transient expression of potential fusion molecules. To identify such a fusion-associated molecule, we established a macrophage fusion assay and generated monoclonal antibodies (mAbs) that alter the fusion of macrophages in vitro. We selected four mAbs that each had the ability to block the fusion but not the aggregation of macrophages in vitro. All four antibodies recognize surface proteins of 150 kDa. The expression of the antigens recognized by all four mAbs is restricted to macrophages that have been induced to fuse in vitro and in vivo and is inducible, transient, and regulated, as neither nonfusing macrophages nor macrophages fused in vitro express these antigens. These results support the hypothesis that macrophage fusion is mediated by specific fusion/adhesion molecules and also provide a means to study the molecular mechanisms of macrophage fusion.

Red/far-red and blue light-responsive regions of maize rbcS-m3 are active in bundle sheath and mesophyll cells, respectively.

Leaves of the C4 plant maize have two major types of photosynthetic cells: a ring of five large bundle sheath cells (BSC) surrounds each vascular bundle and smaller mesophyll cells (MC) lie between the cylinders of bundle sheath cells. The enzyme ribulose bisphosphate carboxylase/oxygenase is encoded by nuclear rbcS and chloroplast rbcL genes. It is not present in MC but is abundant in adjacent BSC of green leaves. As reported previously, the separate regions of rbcS-m3, which are required for stimulating transcription of the gene in BSC and for suppressing expression of reporter genes in MC, were identified by an in situ expression assay; expression was not suppressed in MC until after leaves of dark-grown seedlings had been illuminated for 24 h. Now we have found that transient expression of rbcS-m3 reporter genes is stimulated in BSC via a red/far-red reversible phytochrome photoperception and signal transduction system but that blue light is required for suppressing rbcS-m3 reporter gene expression in MC. Blue light is also required for the suppression system to develop in MC. Thus, the maize gene rbcS-m3 contains certain sequences that are responsive to a phytochrome photoperception and signal transduction system and other regions that respond to a UVA/blue light photoperception and signal transduction system. Various models of "coaction" of plant photoreceptors have been advanced; these observations show the basis for one type of coaction.

Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor.

A G protein-coupled receptor for the pineal hormone melatonin was recently cloned from mammals and designated the Mel1a melatonin receptor. We now report the cloning of a second G protein-coupled melatonin receptor from humans and designate it the Mel1b melatonin receptor. The Mel1b receptor cDNA encodes a protein of 362 amino acids that is 60% identical at the amino acid level to the human Mel1a receptor. Transient expression of the Mel1b receptor in COS-1 cells results in high-affinity 2-[125I]iodomelatonin binding (Kd = 160 +/- 30 pM). In addition, the rank order of inhibition of specific 2-[125I]iodomelatonin binding by eight ligands is similar to that exhibited by the Mel1a melatonin receptor. Functional studies of NIH 3T3 cells stably expressing the Mel1b melatonin receptor indicate that it is coupled to inhibition of adenylyl cyclase. Comparative reverse transcription PCR shows that the Mel1b melatonin receptor is expressed in retina and, to a lesser extent, brain. PCR analysis of human-rodent somatic cell hybrids maps the Mel1b receptor gene (MTNR1B) to human chromosome 11q21-22. The Mel1b melatonin receptor may mediate the reported actions of melatonin in retina and participate in some of the neurobiological effects of melatonin in mammals.

High-affinity RNA-binding domains of alfalfa mosaic virus coat protein are not required for coat protein-mediated resistance.

A virus-based vector was used for the transient expression of the alfalfa mosaic virus coat protein (CP) gene in protoplasts and plants. The accumulation of wild-type CP conferred strong protection against subsequent alfalfa mosaic virus infection, enabling the efficacy of CP mutants to be determined without developing transgenic plants. Expression of the CP mRNA alone without CP accumulation conferred weaker protection against infection. The activity of the N-terminal mutant CPs in protection did not correlate with their activities in genome activation. The activity of a C-terminal mutant suggested that encapsidation did not have a role in protection. Our results indicate that interaction of the CP with alfalfa mosaic virus RNA is not important in protection, thereby leaving open the possibility that interactions with host factors lead to protection.

Tissue specificity of a glucocorticoid-dependent enhancer in transgenic mice.

The glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase were associated with the regulatory sequences of a cellular gene expressed ubiquitously--that coding for the largest subunit of RNA polymerase II. In transient expression assays, glucocorticoid responsiveness of the hybrid regulatory regions depends on the spatial relationship and number of regulatory elements. Two parameters affect the ratio of induction by glucocorticoids: the basal level of the hybrid promoter that is affected by the RNA polymerase II regulatory sequences and the glucocorticoid-induced level that depends on the distance between the GRUs and the TATA box. A fully active glucocorticoid-responsive hybrid gene was used to generate transgenic mice. Results show that a composite regulatory pattern is obtained: ubiquitous basal expression characteristic of the RNA polymerase II gene and liver-specific glucocorticoid activation characteristic of the tyrosine aminotransferase GRUs. This result demonstrates that the activity of the tyrosine aminotransferase GRUs is cell-type-specific not only in cultured cells but also in the whole animal.

Transcriptional activation of human adult alpha-globin genes by hypersensitive site-40 enhancer: function of nuclear factor-binding motifs occupied in erythroid cells.

The developmental stage- and erythroid lineage-specific activation of the human embryonic zeta- and fetal/adult alpha-globin genes is controlled by an upstream regulatory element [hypersensitive site (HS)-40] with locus control region properties, a process mediated by multiple nuclear factor-DNA complexes. In vitro DNase I protection experiments of the two G+C-rich, adult alpha-globin promoters have revealed a number of binding sites for nuclear factors that are common to HeLa and K-562 extracts. However, genomic footprinting analysis has demonstrated that only a subset of these sites, clustered between -130 and +1, is occupied in an erythroid tissue-specific manner. The function of these in vivo-occupied motifs of the alpha-globin promoters, as well as those previously mapped in the HS-40 region, is assayed by site-directed mutagenesis and transient expression in embryonic/fetal erythroid K-562 cells. These studies, together with our expression data on the human embryonic zeta-globin promoter, provide a comprehensive view of the functional roles of individual nuclear factor-DNA complexes in the final stages of transcriptional activation of the human alpha-like globin promoters by the HS-40 element.

Cloning of a receptor for amphibian [Phe13]bombesin distinct from the receptor for gastrin-releasing peptide: identification of a fourth bombesin receptor subtype (BB4).

Bombesin is a tetradecapeptide originally isolated from frog skin and demonstrated to have a wide range of actions in mammals. Based on structural homology and similar biological activities, gastrin-releasing peptide (GRP) has been considered the mammalian equivalent of bombesin. We previously reported that frogs have both GRP and bombesin, which therefore are distinct peptides. We now report the cloning of a bombesin receptor subtype (BB4) that has higher affinity for bombesin than GRP. PCR was used to amplify cDNAs related to the known bombesin receptors from frog brain. Sequence analysis of the amplified cDNAs revealed 3 classes of receptor subtypes. Based on amino acid homology, two classes were clearly the amphibian homologs of the GRP and neuromedin B receptors. The third class was unusual and a full-length clone was isolated from a Bombina orientalis brain cDNA library. Expression of the receptor in Xenopus oocytes demonstrated that the receptor responded to picomolar concentrations of [Phe13]-bombesin, the form of bombesin most prevalent in frog brain. The relative rank potency of bombesin-like peptides for this receptor was [Phe13]bombesin > [Leu13]bombesin > GRP > neuromedin B. In contrast, the rank potency for the GRP receptor is GRP > [Leu13]bombesin > [Phe13]bombesin > neuromedin B. Transient expression in CHOP cells gave a Ki for [Phe13]bombesin of 0.2 nM versus a Ki of 2.1 nM for GRP. Distribution analysis showed that this receptor was expressed only in brain, consistent with the distribution of [Phe13]-bombesin. Thus, based on distribution and affinity, this bombesin receptor is the receptor for [Phe13]bombesin. Phylogenetic analysis suggests that this receptor separated prior to separation of the GRP and neuromedin B receptors; thus, BB4 receptors and their cognate ligands may also exist in mammals.

Functional interactions between the retinoblastoma (Rb) protein and Sp-family members: superactivation by Rb requires amino acids necessary for growth suppression.

The transient expression of the retinoblastoma protein (Rb) regulates the transcription of a variety of growth-control genes, including c-fos, c-myc, and the gene for transforming growth factor beta 1 via discrete promoter sequences termed retinoblastoma control elements (RCE). Previous analyses have shown that Sp1 is one of three RCE-binding proteins identified in nuclear extracts and that Rb functionally interacts with Sp1 in vivo, resulting in the "superactivation" of Sp1-mediated transcription. By immunochemical and biochemical criteria, we report that an Sp1-related transcription factor, Sp3, is a second RCE-binding protein. Furthermore, in transient cotransfection assays, we report that Rb "superactivates" Sp3-mediated RCE-dependent transcription in vivo and that levels of superactivation are dependent on the trans-activator (Sp1 or Sp3) studied. Using expression vectors carrying mutated Rb cDNAs, we have identified two portions of Rb required for superactivation: (i) a portion of the Rb "pocket" (amino acids 614-839) previously determined to be required for physical interactions between Rb and transcription factors such as E2F-1 and (ii) a novel amino-terminal region (amino acids 140-202). Since both of these regions of Rb are targets of mutation in human tumors, our data suggest that superactivation of Sp1/Sp3 may play a role in Rb-mediated growth suppression and/or the induction of differentiation.

Dissection of miRNA Pathways Using Arabidopsis Mesophyll Protoplasts

MicroRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (he) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG(4) Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine-mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of he genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant he and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra- and extracellular ratios of HC and LC polypeptides for three GS-CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra- and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

Ebola virus glycoprotein GP is not cytotoxic when expressed constitutively at a moderate level

Transient expression of Ebola virus (EBOV) glycoprotein GP causes downregulation of surface proteins, cell rounding and detachment, a phenomenon believed to play a central role in the pathogenicity of the virus. In this study, evidence that moderate expression of GP does not result in such morphological changes was provided. It was shown that GP continuously produced in 293T cells from the Kunjin virus replicon was correctly processed and transported to the plasma membrane without affecting the surface expression of beta 1 and alpha 5 integrins and major histocompatibility complex I molecules. The level of GIP expression in Kunjin replicon GP-expressing cells was similar to that observed in cells infected with EBOV early in infection and lower than that produced in cells transfected with plasmid DNA, phCMV-GP(1) expressing GP from a strong promoter. Importantly, transient transfection of Kunjin replicon GIP-expressing cells with GIP-coding plasmid DNA resulted in overexpression of GP, which lead to the downregulation of surface molecules and massive rounding and detachment of transfected cells. Here, it was also demonstrated that cell rounding and downregulation of the surface markers are the late events in EBOV infection, whereas synthesis and massive release of virus particles occur at early steps and do not cause significant cytotoxic effects. These findings indicate that the synthesis of EBOV GP in virus-infected cells is controlled well by several mechanisms that do not allow GP overexpression and hence the early appearance of its cytotoxic properties.

Regulation of bone biology by prostaglandin endoperoxide H synthases (PGHS): A rose by any other name...

It is well established that prostaglandins are essential mediators of bone resorption and formation. In the early 1990s, it was discovered that enzymatic reactions producing prostaglandins were regulated by two cyclooxygenase enzymes, one producing prostaglandins constitutively in tissues like the stomach, prostaglandin endoperoxide H synthase-1 (PGHS-1 or COX-1), and another induced by mitogens or inflammatory mediators (PGHS-2 or COX-2). This neat distinction has not been maintained because both enzymes act in different cell systems to provide physiological signaling, constitutively or by induction under certain conditions. For example, the regulation patterns of PGHS-1 and PGHS-2 are distinct, but the evidence shows that PGHS-2 functions constitutively in the skeleton. PGHS-2 hits quickly been established, therefore, as a key regulator of bone biology, capable of rapid and transient expression in bone cells, and mediating osteoclastogenesis, mechanotransduction, bone formation and fracture repair. The goal of this review is to Summarize the current state of our knowledge of PGHS regulation of bone metabolism and to identify some of the key unresolved challenges and questions that require further study. (c) 2006 Elsevier Ltd. All rights reserved.

Production d'IgG sialylées en CHO et impact sur leurs fonctions effectrices

La sialylation des N-glycanes du fragment Fc des immunogobulines G (IgG) est une modification peu fréquente des IgG humaines. Pourtant, elle est l’objet de beaucoup d’attention depuis que deux articles fondateurs ont été publiés, qui montrent l’un que la sialylation des IgG diminue leur capacité à déclencher la cytotoxicité cellulaire dépendant de l’anticorps (ADCC), et l’autre que les IgG sialylées en α2,6 seraient la fraction efficace des IgG intraveineuses (IgIV) anti-inflammatoires. Les anticorps monoclonaux thérapeutiques, qui sont le plus souvent des IgG recombinantes produites en culture de cellules de mammifère, connaissent depuis la fin des années 90 un succès et une croissance phénoménaux sur le marché pharmaceutique. La maîtrise de la N-glycosylation du Fc des IgG est une clé de l’efficacité des anticorps monoclonaux. Si les IgG sialylées sont des molécules peu fréquentes in vivo, elles sont très rares en culture cellulaire. Dans cette étude, nous avons développé une méthode de production d’IgG avec une sialylation de type humain en cellules CHO. Nous avons travaillé principalement sur la mise au point d’une stratégie de production d’IgG sialylées par co-expression transitoire d’une IgG1 avec la β1,4-galactosyltransférase I (β4GTI) et la β-galactoside-α2,6-sialyltransférase I (ST6GalI). Nous avons montré que cette méthode permettait d’enrichir l’IgG1 en glycane fucosylé di-galactosylé mono-α2,6-sialylé G2FS(6)1, qui est le glycane sialylé présent sur les IgG humaines. Nous avons ensuite adapté cette méthode à la production d’IgG présentant des profils de glycosylation riches en acides sialiques, riches en galactose terminal, et/ou appauvris en fucosylation. L’analyse des profils de glycosylation obtenus par la co-expression de diverses combinaisons enzymatiques avec l’IgG1 native ou une version mutante de l’IgG1 (F243A), a permis de discuter des influences respectives de la sous-galactosylation des IgG1 en CHO et des contraintes structurales du Fc dans la limitation de la sialylation des IgG en CHO. Nous avons ensuite utilisé les IgG1 produites avec différents profils de glycosylation afin d’évaluer l’impact de la sialylation α2,6 sur l’interaction de l’IgG avec le récepteur FcγRIIIa, principal récepteur impliqué dans la réponse ADCC. Nous avons montré que la sialylation α2,6 augmentait la stabilité du complexe formé par l’IgG avec le FcγRIIIa, mais que ce bénéfice n’était pas directement traduit par une augmentation de l’efficacité ADCC de l’anticorps. Enfin, nous avons débuté le développement d’une plateforme d’expression stable d’IgG sialylées compatible avec une production à l’échelle industrielle. Nous avons obtenu une lignée capable de produire des IgG enrichies en G2FS(6)1 à hauteur de 400 mg/L. Cette étude a contribué à une meilleure compréhension de l’impact de la sialylation sur les fonctions effectrices des IgG, et a permis d’augmenter la maîtrise des techniques de modulation du profil de glycosylation des IgG en culture cellulaire.

Rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293

La possibilité de programmer une cellule dans le but de produire une protéine d’intérêt est apparue au début des années 1970 avec l’essor du génie génétique. Environ dix années plus tard, l’insuline issue de la plateforme de production microbienne Escherichia coli, fut la première protéine recombinante (r-protéine) humaine commercialisée. Les défis associés à la production de r-protéines plus complexes et glycosylées ont amené l’industrie biopharmaceutique à développer des systèmes d’expression en cellules de mammifères. Ces derniers permettent d’obtenir des protéines humaines correctement repliées et de ce fait, biologiquement actives. Afin de transférer le gène d’intérêt dans les cellules de mammifères, le polyéthylènimine (PEI) est certainement un des vecteurs synthétiques le plus utilisé en raison de son efficacité, mais aussi sa simplicité d’élaboration, son faible coût et sa stabilité en solution qui facilite son utilisation. Il est donc largement employé dans le contexte de la production de r-protéines à grande échelle et fait l’objet d’intenses recherches dans le domaine de la thérapie génique non virale. Le PEI est capable de condenser efficacement l’ADN plasmidique (vecteur d’expression contenant le gène d’intérêt) pour former des complexes de petites tailles appelés polyplexes. Ces derniers doivent contourner plusieurs étapes limitantes afin de délivrer le gène d’intérêt au noyau de la cellule hôte. Dans les conditions optimales du transfert de gène par le PEI, les polyplexes arborent une charge positive nette interagissant de manière électrostatique avec les protéoglycanes à héparane sulfate (HSPG) qui décorent la surface cellulaire. On observe deux familles d’HSPG exprimés en abondance à la surface des cellules de mammifères : les syndécanes (4 membres, SDC1-4) et les glypicanes (6 membres, GPC1-6). Si l’implication des HSPG dans l’attachement cellulaire des polyplexes est aujourd’hui largement acceptée, leur rôle individuel vis-à-vis de cet attachement et des étapes subséquentes du transfert de gène reste à confirmer. Après avoir optimisées les conditions de transfection des cellules de mammifères CHO et HEK293 dans le but de produire des r-protéines secrétées, nous avons entrepris des cinétiques de capture, d’internalisation des polyplexes et aussi d’expression du transgène afin de mieux comprendre le processus de transfert de gène. Nous avons pu observer des différences au niveau de ces paramètres de transfection dépendamment du système d’expression et des caractéristiques structurelles du PEI utilisé. Ces résultats présentés sous forme d’articles scientifiques constituent une base solide de l’enchaînement dans le temps des évènements essentiels à une transfection efficace des cellules CHO et HEK293 par le PEI. Chaque type cellulaire possède un profil d’expression des HSPG qui lui est propre, ces derniers étant plus ou moins permissifs au transfert de gène. En effet, une étude menée dans notre laboratoire montre que les SDC1 et SDC2 ont des rôles opposés vis-à-vis du transfert de gène. Alors que tous deux sont capables de lier les polyplexes, l’expression de SDC1 permet leur internalisation contrairement à l’expression de SDC2 qui l’inhibe. De plus, lorsque le SDC1 est exprimé à la surface des cellules HEK293, l’efficacité de transfection est augmentée de douze pourcents. En utilisant la capacité de SDC1 à induire l’internalisation des polyplexes, nous avons étudié le trafic intracellulaire des complexes SDC1 / polyplexes dans les cellules HEK293. De plus, nos observations suggèrent une nouvelle voie par laquelle les polyplexes pourraient atteindre efficacement le noyau cellulaire. Dans le contexte du transfert de gène, les HSPG sont essentiellement étudiés dans leur globalité. S’il est vrai que le rôle des syndécanes dans ce contexte est le sujet de quelques études, celui des glypicanes est inexploré. Grâce à une série de traitements chimiques et enzymatiques visant une approche « perte de fonction », l’importance de la sulfatation comme modification post-traductionnelle, l’effet des chaînes d’héparanes sulfates mais aussi des glypicanes sur l’attachement, l’internalisation des polyplexes, et l’expression du transgène ont été étudiés dans les cellules CHO et HEK293. L’ensemble de nos observations indique clairement que le rôle des HSPG dans le transfert de gène devrait être investigué individuellement plutôt que collectivement. En effet, le rôle spécifique de chaque membre des HSPG sur la capture des polyplexes et leur permissivité à l’expression génique demeure encore inconnu. En exprimant de manière transitoire chaque membre des syndécanes et glypicanes à la surface des cellules CHO, nous avons déterminé leur effet inhibiteur ou activateur sur la capture des polyplexes sans pouvoir conclure quant à l’effet de cette surexpression sur l’efficacité de transfection. Par contre, lorsqu’ils sont présents dans le milieu de culture, le domaine extracellulaire des HSPG réduit l’efficacité de transfection des cellules CHO sans induire la dissociation des polyplexes. Curieusement, lorsque chaque HSPG est exprimé de manière stable dans les cellules CHO, seulement une légère modulation de l’expression du transgène a pu être observée. Ces travaux ont contribué à la compréhension des mécanismes d'action du vecteur polycationique polyéthylènimine et à préciser le rôle des protéoglycanes à héparane sulfate dans le transfert de gène des cellules CHO et HEK293.