High-frequency linkage of co-expressing T-DNA in transgenic Arabidopsis thaliana transformed by vacuum-infiltration of Agrobacterium tumefaciens

The efficiency of co-expression and linkage of distinct T-DNAs present in separate Agrobacterium tumefaciens was analysed in Arabidopsis thaliana transformed by the vacuum infiltration method. Co-expression was monitored by the synthesis of three bacterial proteins involved in the production of polyhydroxybutyrate (PHB) in the plastids. Out of 80 kanamycin-resistant transgenic plants analysed, 13 plants were co-transformed with the two distinct T-DNAs and produced PHB. Of those, 7 lines had a kanamycin-resistance segregation ratio consistent with the presence of a single functional insert. Genetic linkage between the distinct T-DNAs was demonstrated for all 13 PHB-producing lines, while physical linkage between the distinct T-DNAs was shown for 12 out of 13 lines. T-DNAs were frequently linked in an inverted orientation about the left borders. Transformation of A. thaliana by the co-infiltration of two A. tumefaciens containing distinct T-DNAs is, thus, an efficient approach for the integration and expression of several transgenes at a single locus. This approach will facilitate the creation and study of novel metabolic pathways requiring the expression of numerous transgenes.

Structural and functional studies of nonstructural protein 2 of the hepatitis C virus reveal its key role as organizer of virion assembly.

Non-structural protein 2 (NS2) plays an important role in hepatitis C virus (HCV) assembly, but neither the exact contribution of this protein to the assembly process nor its complete structure are known. In this study we used a combination of genetic, biochemical and structural methods to decipher the role of NS2 in infectious virus particle formation. A large panel of NS2 mutations targeting the N-terminal membrane binding region was generated. They were selected based on a membrane topology model that we established by determining the NMR structures of N-terminal NS2 transmembrane segments. Mutants affected in virion assembly, but not RNA replication, were selected for pseudoreversion in cell culture. Rescue mutations restoring virus assembly to various degrees emerged in E2, p7, NS3 and NS2 itself arguing for an interaction between these proteins. To confirm this assumption we developed a fully functional JFH1 genome expressing an N-terminally tagged NS2 demonstrating efficient pull-down of NS2 with p7, E2 and NS3 and, to a lower extent, NS5A. Several of the mutations blocking virus assembly disrupted some of these interactions that were restored to various degrees by those pseudoreversions that also restored assembly. Immunofluorescence analyses revealed a time-dependent NS2 colocalization with E2 at sites close to lipid droplets (LDs) together with NS3 and NS5A. Importantly, NS2 of a mutant defective in assembly abrogates NS2 colocalization around LDs with E2 and NS3, which is restored by a pseudoreversion in p7, whereas NS5A is recruited to LDs in an NS2-independent manner. In conclusion, our results suggest that NS2 orchestrates HCV particle formation by participation in multiple protein-protein interactions required for their recruitment to assembly sites in close proximity of LDs.

Increased audiovisual integration in cochlear-implanted deaf patients: independent components analysis of longitudinal positron emission tomography data.

It has been demonstrated in earlier studies that patients with a cochlear implant have increased abilities for audio-visual integration because the crude information transmitted by the cochlear implant requires the persistent use of the complementary speech information from the visual channel. The brain network for these abilities needs to be clarified. We used an independent components analysis (ICA) of the activation (H2 (15) O) positron emission tomography data to explore occipito-temporal brain activity in post-lingually deaf patients with unilaterally implanted cochlear implants at several months post-implantation (T1), shortly after implantation (T0) and in normal hearing controls. In between-group analysis, patients at T1 had greater blood flow in the left middle temporal cortex as compared with T0 and normal hearing controls. In within-group analysis, patients at T0 had a task-related ICA component in the visual cortex, and patients at T1 had one task-related ICA component in the left middle temporal cortex and the other in the visual cortex. The time courses of temporal and visual activities during the positron emission tomography examination at T1 were highly correlated, meaning that synchronized integrative activity occurred. The greater involvement of the visual cortex and its close coupling with the temporal cortex at T1 confirm the importance of audio-visual integration in more experienced cochlear implant subjects at the cortical level.

Lack of association between the protein tyrosine phosphatase non-receptor type 22 R263Q and R620W functional genetic variants and endogenous non-anterior uveitis.

OBJECTIVE Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility. METHODS We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test. RESULTS Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information. CONCLUSIONS Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.

Structural and functional brain changes in middle-aged type 2 diabetic patients: a cross-sectional study.

BACKGROUND Type 2 diabetes mellitus (T2DM) is an emerging risk factor for cognitive impairment. Whether this impairment is a direct effect of this metabolic disorder on brain function, a consequence of vascular disease, or both, remains unknown. Structural and functional neuroimaging studies in patients with T2DM could help to elucidate this question. OBJECTIVE We designed a cross-sectional study comparing 25 T2DM patients with 25 age- and gender-matched healthy control participants. Clinical information, APOE genotype, lipid and glucose analysis, structural cerebral magnetic resonance imaging including voxel-based morphometry, and F-18 fluorodeoxyglucose positron emission tomography were obtained in all subjects. METHODS Gray matter densities and metabolic differences between groups were analyzed using statistical parametric mapping. In addition to comparing the neuroimaging profiles of both groups, we correlated neuroimaging findings with HbA1c levels, duration of T2DM, and insulin resistance measurement (HOMA-IR) in the diabetic patients group. Results: Patients with T2DM presented reduced gray matter densities and reduced cerebral glucose metabolism in several fronto-temporal brain regions after controlling for various vascular risk factors. Furthermore, within the T2DM group, longer disease duration, and higher HbA1c levels and HOMA-IR were associated with lower gray matter density and reduced cerebral glucose metabolism in fronto-temporal regions. CONCLUSION In agreement with previous reports, our findings indicate that T2DM leads to structural and metabolic abnormalities in fronto-temporal areas. Furthermore, they suggest that these abnormalities are not entirely explained by the role of T2DM as a cardiovascular risk factor.

Two functional variants of IRF5 influence the development of macular edema in patients with non-anterior uveitis.

OBJECTIVE Interferon (IFN) signaling plays a crucial role in autoimmunity. Genetic variation in interferon regulatory factor 5 (IRF5), a major regulator of the type I interferon induction, has been associated with risk of developing several autoimmune diseases. In the current study we aimed to evaluate whether three sets of correlated IRF5 genetic variants, independently associated with SLE and with different functional roles, are involved in uveitis susceptibility and its clinical subphenotypes. METHODS Three IRF5 polymorphisms, rs2004640, rs2070197 and rs10954213, representative of each group, were genotyped using TaqMan® allelic discrimination assays in a total of 263 non-anterior uveitis patients and 724 healthy controls of Spanish origin. RESULTS A clear association between two of the three analyzed genetic variants, rs2004640 and rs10954213, and the absence of macular edema was observed in the case/control analysis (P FDR =5.07E-03, OR=1.48, CI 95%=1.14-1.92 and P FDR =3.37E-03, OR=1.54, CI 95%=1.19-2.01, respectively). Consistently, the subphenotype analysis accordingly with the presence/absence of this clinical condition also reached statistical significance (rs2004640: P=0.037, OR=0.69, CI 95%=0.48-0.98; rs10954213: P=0.030, OR=0.67, CI 95%=0.47-0.96), thus suggesting that both IRF5 genetic variants are specifically associated with the lack of macular edema in uveitis patients. CONCLUSION Our results clearly showed for the first time that two functional genetic variants of IRF5 may play a role in the development of macular edema in non-anterior uveitis patients. Identifying genetic markers for macular edema could lead to the possibility of developing novel treatments or preventive therapies.

Lactotransferrin gene functional polymorphisms do not influence susceptibility to human immunodeficiency virus-1 mother-to-child transmission in different ethnic groups

Lactotransferrin, also known as lactoferrin, is an iron binding glycoprotein that displays antiviral activity against many different infectious agents, including human immunodeficiency virus (HIV)-1. Lactotransferrin is present in the breast milk and in the female genitourinary mucosa and it has been hypothesised as a possible candidate to prevent mother-to-child HIV-1 transmission. To verify if two functional polymorphisms, Thr29Ala and Arg47Lys, in the lactotransferrin encoding gene (LTF) could affect HIV-1 infection and vertical transmission, a preliminary association study was performed in 238 HIV-1 positive and 99 HIV-1 negative children from Brazil, Italy, Africa and India. No statistically significant association for the Thr29Ala and Arg47Lys LTF polymorphisms and HIV-1 susceptibility in the studied populations was found. Additionally LTF polymorphisms frequencies were compared between the four different ethnic groups.

Functional and molecular evolutionary analysis of the "Brevix Radix" gene family in mono-and dicotyledonous plants

ABSTRACTIn contrast to animals, plants cannot move from their place of birth and, therefore, need to adapt to their particular habitat in order to survive. Thus, plant development is remarkably plastic, making plants an ideal system for the isolation of genes that account for intraspecific natural variation and possibly environmental adaptation. However, to date, this approach mostly identified null alleles and missed mutations with subtle effects. For instance, BREVIS RADIX (BRX) has been isolated as a key regulator of root growth through a naturally occurring loss-of-function allele in the Arabidopsis thaliana accession Uk-1 and is the founding member of a highly-conserved plant-specific gene family.In this work, we show that a strong selective pressure is acting on the BRX gene family and dates back before the monocot-dicot divergence. However, functional diversification is observed mainly in dicotyledon BRX family genes and is correlated with acceleration in the evolutionary rates in the N-terminal regions. Population genetic data revealed that BRX is highly conserved across Arabidopsis accessions and presents signatures of adaptation. Interestingly, a seven amino acid deletion polymorphism in BRX sequence was found in a few accessions, which seems to be responsible for their enhanced primary root growth. Nevertheless, BRX might not only be active in the root, as suggested by its expression in the shoot. Indeed, leaves and cotyledons of brx mutants are significantly smaller than wild- type. This phenotype is a direct consequence of the absence of BRX function in the shoot rather than an indirect effect of an altered root system growth. Interestingly, cotyledons of brx plants reflect the same physiological defects as the root. Moreover, phenotypes in BRX gain-of-function plants, such as epinastic leaves and increased epidermal cell size, could be associated with an increase in leaf brassinosteroid content.Collectively, these results indicate that BRX contributes to local adaptation by ubiquitously regulating plant growth, probably through the modulation of brassinosteroid biosynthesis.RÉSUMÉContrairement à la plupart des animaux, les plantes ne peuvent se mouvoir et doivent ainsi s'adapter à leur environnement pour survivre. Pour cette raison, elles représentent un système idéal pour l'identification de gènes contribuant à la variation naturelle intra- spécifique, ainsi qu'à l'adaptation. Cependant, cette approche a, jusqu'à présent, surtout permis d'isoler des allèles nuls et non des mutations conférant des effets plus subtiles. C'est le cas du gène Β REVIS RADIX (BRX), un régulateur clé de la croissance racinaire, qui a été identifié grâce à un allèle non-fonctionnel présent dans l'accession naturelle d'Arabidopsis thaliana Uk-1. BRX et ses homologues des plantes mono- et dicotylédones forment une famille très conservée et spécifique aux plantes.Dans ce travail, nous démontrons que la famille de gènes BRX est soumise à une forte pression de sélection qui remonte avant la divergence entre mono- et dicotylédones. Cependant, une diversification fonctionnelle a été observée chez les gènes des dicotylédones et corrèle avec une accélération de la vitesse d'évolution dans leur région N- terminale. Une analyse génétique de différentes accessions naturelles d'Arabidopsis a révélé que BRX est hautement conservé et présente des signatures d'adaptation. Remarquablement, un polymorphisme de délétion de sept acides aminés a été détecté dans quelques accessions et a pour conséquence une plus forte croissance de la racine primaire. Néanmoins, il semble que le rôle de BRX ne se limite pas qu'à la racine, comme indiqué par son expression dans les parties aériennes de la plante. En effet, les mutants brx présentent des cotylédons et des feuilles significativement plus petits que le type sauvage, une conséquence directe de l'absence d'activité de BRX dans ces organes. Nous avons aussi noté que les cotylédons des mutants brx, à l'instar des racines, ont une perception altérée de l'auxine et peuvent être complémentés par l'application exogène de brassinostéroïdes. De plus, dans des plantes présentant un gain de fonction BRX, les feuilles sont épinastiques et les cellules de leur épiderme plus grandes. Ces phénotypes sont accompagnés d'une augmentation de la concentration de brassinostéroïdes dans les feuilles. Conjointement, ces résultats démontrent que BRX contribue à une adaptation locale de la plante par la régulation générale de sa croissance, probablement en modulant la biosynthèse des brassinostéroïdes.

Functional MRI Mapping of the Human Auditory Cortex

Mapping the human auditory cortex with standard functional imaging techniques is difficult because of its small size and angular position along the Sylvian fissure. As a result, the exact number and location of auditory cortex areas in the human remains unknown. In a first experiment, we measured the two largest tonotopic areas of primary auditory cortex (PAC, Al and R) using high-resolution functional MRI at 7 Tesla relative to the underlying anatomy of Heschl's gyrus (HG). The data reveals a clear anatomical- functional relationship that indicates the location of PAC across the range of common morphological variants of HG (single gyri, partial duplication and complete duplication). Human PAC tonotopic areas are oriented along an oblique posterior-to-anterior axis with mirror-symmetric frequency gradients perpendicular to HG, as in the macaque. In a second experiment, we tested whether these primary frequency-tuned units were modulated by selective attention to preferred vs. non-preferred sound frequencies in the dynamic manner needed to account for human listening abilities in noisy environments, such as cocktail parties or busy streets. We used a dual-stream selective attention experiment where subjects attended to one of two competing tonal streams presented simultaneously to different ears. Attention to low-frequency tones (250 Hz) enhanced neural responses within low-frequency-tuned voxels relative to high (4000 Hz), and vice versa when at-tention switched from high to low. Human PAC is able to tune into attended frequency channels and can switch frequencies on demand, like a radio. In a third experiment, we investigated repetition suppression effects to environmental sounds within primary and non-primary early-stage auditory areas, identified with the tonotopic mapping design. Repeated presentations of sounds from the same sources, as compared to different sources, gave repetition suppression effects within posterior and medial non-primary areas of the right hemisphere, reflecting their potential involvement in semantic representations. These three studies were conducted at 7 Tesla with high-resolution imaging. However, 7 Tesla scanners are, for the moment, not yet used for clinical diagnosis and mostly reside in institutions external to hospitals. Thus, hospital-based clinical functional and structural studies are mainly performed using lower field systems (1.5 or 3 Tesla). In a fourth experiment, we acquired tonotopic maps at 3 and 7 Tesla and evaluated the consistency of a tonotopic mapping paradigm between scanners. Mirror-symmetric gradients within PAC were highly similar at 7 and 3 Tesla across renderings at different spatial resolutions. We concluded that the tonotopic mapping paradigm is robust and suitable for definition of primary tonotopic areas, also at 3 Tesla. Finally, in a fifth study, we considered whether focal brain lesions alter tonotopic representations in the intact ipsi- and contralesional primary auditory cortex in three patients with hemispheric or cerebellar lesions, without and with auditory complaints. We found evidence for tonotopic reorganisation at the level of the primary auditory cortex in cases of brain lesions independently of auditory complaints. Overall, these results reflect a certain degree of plasticity within primary auditory cortex in different populations of subjects, assessed at different field strengths. - La cartographie du cortex auditif chez l'humain est difficile à réaliser avec des techniques d'imagerie fonctionnelle standard, étant donné sa petite taille et position angulaire le long de la fissure sylvienne. En conséquence, le nombre et l'emplacement exacts des différentes aires du cortex auditif restent inconnus chez l'homme. Lors d'une première expérience, nous avons mesuré, avec de l'imagerie par résonance magnétique à haute intensité (IRMf à 7 Tesla) chez des sujets humains sains, deux larges aires au sein du cortex auditif primaire (PAC; Al et R) avec une représentation spécifique des fréquences pures préférées - ou tonotopie. Nos résultats ont démontré une relation anatomico- fonctionnelle qui définit clairement la position du PAC à travers toutes les variantes du gyrus d'Heschl's (HG). Les aires tonotopiques du PAC humain sont orientées le long d'un axe postéro-antérieur oblique avec des gradients de fréquences spécifiques perpendiculaires à HG, d'une manière similaire à celles mesurées chez le singe. Dans une deuxième expérience, nous avons testé si ces aires primaires pouvaient être modulées, de façon dynamique, par une attention sélective pour des fréquences préférées par rapport à celles non-préférées. Cette modulation est primordiale lors d'interactions sociales chez l'humain en présence de bruits distracteurs tels que d'autres discussions ou un environnement sonore nuisible (comme par exemple, dans la circulation routière). Dans cette étude, nous avons utilisé une expérience d'attention sélective où le sujet devait être attentif à une des deux voies sonores présentées simultanément à chaque oreille. Lorsque le sujet portait était attentif aux sons de basses fréquences (250 Hz), la réponse neuronale relative à ces fréquences augmentait par rapport à celle des hautes fréquences (4000 Hz), et vice versa lorsque l'attention passait des hautes aux basses fréquences. De ce fait, nous pouvons dire que PAC est capable de focaliser sur la fréquence attendue et de changer de canal selon la demande, comme une radio. Lors d'une troisième expérience, nous avons étudié les effets de suppression due à la répétition de sons environnementaux dans les aires auditives primaires et non-primaires, d'abord identifiées via le protocole de la première étude. La présentation répétée de sons provenant de la même source sonore, par rapport à de sons de différentes sources sonores, a induit un effet de suppression dans les aires postérieures et médiales auditives non-primaires de l'hémisphère droite, reflétant une implication de ces aires dans la représentation de la catégorie sémantique. Ces trois études ont été réalisées avec de l'imagerie à haute résolution à 7 Tesla. Cependant, les scanners 7 Tesla ne sont pour le moment utilisés que pour de la recherche fondamentale, principalement dans des institutions externes, parfois proches du patient mais pas directement à son chevet. L'imagerie fonctionnelle et structurelle clinique se fait actuellement principalement avec des infrastructures cliniques à 1.5 ou 3 Tesla. Dans le cadre dune quatrième expérience, nous avons avons évalués la cohérence du paradigme de cartographie tonotopique à travers différents scanners (3 et 7 Tesla) chez les mêmes sujets. Nos résultats démontrent des gradients de fréquences définissant PAC très similaires à 3 et 7 Tesla. De ce fait, notre paradigme de définition des aires primaires auditives est robuste et applicable cliniquement. Finalement, nous avons évalués l'impact de lésions focales sur les représentations tonotopiques des aires auditives primaires des hémisphères intactes contralésionales et ipsilésionales chez trois patients avec des lésions hémisphériques ou cérébélleuses avec ou sans plaintes auditives. Nous avons trouvé l'évidence d'une certaine réorganisation des représentations topographiques au niveau de PAC dans le cas de lésions cérébrales indépendamment des plaintes auditives. En conclusion, nos résultats démontrent une certaine plasticité du cortex auditif primaire avec différentes populations de sujets et différents champs magnétiques.

Dissecting TCR-MHC/peptide complex interactions with A2/peptide multimers incorporating tumor antigen peptide variants: crucial role of interaction kinetics on functional outcomes.

Soluble peptide/MHC-class-I (pMHC) multimers have recently emerged as unique reagents for the study of specific interactions between the pMHC complex and the TCR. Here, we assessed the relative binding efficiency of a panel of multimers incorporating single-alanine-substituted variants of the tumor-antigen-derived peptide MAGE-A10(254-262) to specific CTL clones displaying different functional avidity. For each individual clone, the efficiency of binding of multimers incorporating MAGE-A10 peptide variants was, in most cases, in good although not linear correlation with the avidity of recognition of the corresponding variant. In addition, we observed two types of discrepancies between efficiency of recognition and multimer binding. First, for some peptide variants, efficient multimer binding was detected in the absence of measurable effector functions. Some of these peptide variants displayed antagonist activity. Second, when comparing different clones we found clear discrepancies between the dose of peptide required to obtain half-maximal lysis in CTL assays and the binding efficiency of the corresponding multimers. These discrepancies, however, were resolved when the differential stability of the TCR/pMHC complexes was determined. For individual clones, decreased recognition correlated with increased TCR/pMHC off-rate. TCR/pMHC complexes formed by antagonist ligands displayed off-rates faster than those of TCR/pMHC complexes formed with weak agonists. In addition, when comparing different clones, the efficiency of multimer staining correlated better with relative multimer off-rates than with half-maximal lysis values. Altogether, the data presented here reconcile and extend our previous results on the impact of the kinetics of interaction of TCR with pMHC complexes on multimer binding and underline the crucial role of TCR/pMHC off-rates for the functional outcome of such interactions.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

Insulated retroviral vectors towards safe and efficient genetic modification of stem cells

In otherwise successful gene therapy trials for the treatment of SCID patients and others, insertional mutagenesis has resulted in leukemia development. Besides the integration of vectors that including strong enhancers, more recently, SIN-vectors have been shown to partially retain oncogenic potential. The identification of genetic elements which would both prevent such activation effects and shield the transgene from silencing, is a main challenge. Previous attempts met with difficulties in producing the vectors and poor efficacy of the insulators (GIE). The improvement of integrating vectors safety has been investigated using new candidate synthetic GIEs. The latter have been introduced in retroviral and lentiviral vectors. Native LTRs, SIN-LTRs, and SIN-insulated constructs have been designed and compared, using two sets of internal promoter, i.e. strong and housekeeping. We could establish that a specific insulator translates at best into functional activity and boundary effect in both vector types. We could also determine that other genetic elements are key determinants in order to achieve accurate expression and viral titre, from these insulated vectors. A dramatic shift in the expression profile is observed in target cells, with a homogenous pattern including data on both cell-lines and primary HSCs from cord blood. The assessment of potential genotoxicity will be presented, based on the comparison of the integration patterns ingenuity in human target cells sampled over a three months period with both reference LTRs and SIN versus test insulated vectors, using high-throughput pyro-sequencing.