At least 10% of spontaneous chromosomal antibiotic resistant mutants of bacteria express a strain-dependent graded reduction of virulence; this correlates linearly with a prolonged generation time. Occasionally, these mutants are temperature sensitive or/and auxotrophe. The work described in this paper provides evidence that in such strains the resistance and the accompanying markers exist only as a functional genetic unit. In a series of transduction experiments with a pathogenic strain of Salmonella typhimurium, it was found that without exception, the resistance and the additional markers were 100% simultaneously transferred. Furthermore, antibiotic-resistant Escherichia coli mutants with prolonged generation time, were isolated from faecal samples; it is thus indicated that, such innocuous mutants occur at any time in the intestine. It is concluded that concerns connecting such mutants to the possibility of resistance dissemination are unfounded; furthermore, even if transfer of resistance ...

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The transduction efficiency can be measured by assessing the fraction of cells expressing TurboGFP or TurboRFP after transduction. This can be achieved by simple microscopic observation using a fluorescence microscope or by FACS analysis 48-72 hours after transduction ...

Finden Sie alle Bücher von Michael D. Mendenhall, Robert C. Dickson - Signal Transduction Protocols. Bei der Büchersuchmaschine eurobuch.com können Sie antiquarische und Neubücher VERGLEICHEN UND SOFORT zum Bestpreis bestellen. 1617374482

Specialized transduction occurs only in some temperate phages. But specialized transduction is an extremely efficient gene transfer mechanism.. In some occasions, DNA from a specific region of the host chromosome is integrated directly into the virus genome-usually replacing some viral genes. The resulting defective transducing phage (temperate phage) particles now have bacterial DNA as a part of genome.. To understand the process of specialized transduction, you must first be aware about lytic cycle of Bacteriophage.. Lets contrast between normal lysogenic cycle and mechanism of transduction. ...

אנו מתארים פרוטוקולים עבור הערכת מידת התמרה על ידי תאי-חודר פפטידים ניצול מערכות הדמיה vivo ex ולאחר מכן הטבעה פרפין, ומיקרוסקופית...

Large collection of high quality biology pictures, photos, images, illustrations, diagrams and posters on marine biology, cell biology, microbiology... for educational purposes.. ...

Large collection of high quality biology pictures, photos, images, illustrations, diagrams and posters on marine biology, cell biology, microbiology... for educational purposes.. ...

The Determination of Importance of Sequences Neighboring the Psi Sequence in Lentiviral Vector Transduction and Packaging Efficiency. . Biblioteca virtual para leer y descargar libros, documentos, trabajos y tesis universitarias en PDF. Material universiario, documentación y tareas realizadas por universitarios en nuestra biblioteca. Para descargar gratis y para leer online.

TY - JOUR. T1 - Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity. AU - Paulk, Nicole K.. AU - Pekrun, Katja. AU - Zhu, Erhua. AU - Nygaard, Sean. AU - Li, Bin. AU - Xu, Jianpeng. AU - Chu, Kirk. AU - Leborgne, Christian. AU - Dane, Allison P.. AU - Haft, Annelise. AU - Zhang, Yue. AU - Zhang, Feijie. AU - Morton, Chris. AU - Valentine, Marcus B.. AU - Davidoff, Andrew M.. AU - Nathwani, Amit C.. AU - Mingozzi, Federico. AU - Grompe, Markus. AU - Alexander, Ian E.. AU - Lisowski, Leszek. AU - Kay, Mark A.. PY - 2017. Y1 - 2017. N2 - Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity ...

A retroviral vector system based on the human immunodeficiency virus (HIV) was developed that, in contrast to a murine leukemia virus-based counterpart, transduced heterologous sequences into HeLa cells and rat fibroblasts blocked in the cell cycle, as well as into human primary macrophages. Additionally, the HIV vector could mediate stable in vivo gene transfer into terminally differentiated neurons. The ability of HIV-based viral vectors to deliver genes in vivo into nondividing cells could increase the applicability of retroviral vectors in human gene therapy.. ...

TY - JOUR. T1 - In vivo stable transduction of humanized liver tissue in chimeric mice via high-capacity adenovirus-lentivirus hybrid vector. AU - Kubo, Shuji. AU - Kataoka, Miho. AU - Tateno, Chise. AU - Yoshizato, Katsutoshi. AU - Kawasaki, Yoshiko. AU - Kimura, Takahiro. AU - Faure-Kumar, Emmanuelle. AU - Palmer, Donna J.. AU - Ng, Philip. AU - Okamura, Haruki. AU - Kasahara, Noriyuki. PY - 2010/1/1. Y1 - 2010/1/1. N2 - We developed hybrid vectors employing high-capacity adenovirus as a first-stage carrier encoding all the components required for in situ production of a second-stage lentivirus, thereby achieving stable transgene expression in secondary target cells. Such vectors have never previously been tested in normal tissues, because of the scarcity of suitable in vivo systems permissive for second-stage lentivirus assembly. Here we employed a novel murine model in which endogenous liver tissue is extensively reconstituted with engrafted human hepatocytes, and successfully achieved ...

We and others have now shown that transduced primary Ag-specific CD4+ T cells provide many beneficial features for targeted immunotherapy (58). Retrovirally transduced cells were easily selected for dose of transgene based on reporter gene expression and were then safely transferred to recipient animals for local product delivery. Although pGC retroviral vectors use a constitutive promoter, in vitro analysis has demonstrated that transgene expression correlates with the activation state of the CD4+ T cell (G. L. Costa and J. M. Benson, unpublished observations). Therefore, it is reasonable to conclude that transduced T cells require Ag presentation in vivo to produce transgenic proteins. This was evident in bioluminescent cell trafficking studies (Fig. 4⇑A), in which luciferase production was no longer detected in naive mice once transduced T cells returned to a quiescent state (9 days after transfer). Interestingly, we observed that MBP-specific hybridomas were more efficient in reducing EAE ...

A mathematical model for nonrandom generalized transduction is proposed and analyzed. The model takes into account the finite number of transducing particle classes for any given marker. The equations for estimation of the distance between markers from cotransduction frequency data are derived and standard errors of the estimates are given. The obtained relationships depend significantly on the number of classes of transducing fragments. The model was applied to estimate the number of transducing fragment classes for a given marker in transduction with phage P22 of Salmonella typhimurium. It was found that the literature data on frequencies of cotransduction in crosses with mutual substitution of selective and nonselective markers can be rationalized most accurately by assuming that the mean number of classes is equal to 2. An improved method for analysis of cotransduction data is proposed on the basis of our model and the results of calculation. The method relies on solving a set of algebraic ...

Investigation of the role of target cell factors in retrovirus transduction. . Biblioteca virtual para leer y descargar libros, documentos, trabajos y tesis universitarias en PDF. Material universiario, documentación y tareas realizadas por universitarios en nuestra biblioteca. Para descargar gratis y para leer online.

Because of the increased efficiency of transduction and expression, and the possible decreased potential for adverse effects, such as insertional mutagenesis compared to retroviruses, we used lentiviral-vector technology to express the fusion constructs in primary human T cells using clinically validated techniques (15). The cDNA sequences containing the various fusion constructions were cloned into a third-generation lentiviral vector in which the CMV promoter was replaced with the EF-1α promoter (16). Lentiviral vector supernatants transduced primary T cells with high efficiency (Fig. 1B). In preliminary experiments, the SS1-transduced T cells were found to have sustained proliferation in the presence of various cell lines that express mesothelin, while culture in the absence of mesothelin-expressing feeder cells failed to sustain T-cell proliferation (data not shown). All constructs were brightly expressed on the surface of CD4 and CD8 T cells, and expression was stable for at least 2 months ...

Generalized transduction is commonly used to move transposon-induced mutations among bacterial strains by selecting for inheritance of a transposonencoded resistance determinant. Although complete cotransduction of the resistance determinant and the chromosomal mutation might be expected, it is often found that when Tn5(Kan) insertion mutations are transduced by bacteriophage P1 most of the nonmutant kanamycin-resistant transductants are due to specialized transduction of Tn5. Such P1::Tn5 specialized transducing phage are not found when a mutant Tn5 element lacking a functional transposase is employed.. ...

Herpes simplex virus type 1 (HSV-1) amplicon preparations are usually quantified as transducing units/ml (TU/ml), with little information on genomic copy/TU ratios. In the present study, two HSV-1 amplicons expressing enhanced green fluorescent protein (EGFP) were analysed by quantitative PCR (qPCR) and transducing activity to obtain genomic copy/TU ratios. One vector (pHSV-GL) contains the HSV-1 packaging signal (pac) and origin of replication (oriS) and the other (pHSV/EBV-GL) includes Epstein-Barr virus (EBV) episomal maintenance elements. The pHSV-GL and pHSV/EBV-GL amplicons were prepared at titres of 7.55x10(7) and 7.24x10(7)TU/ml, containing 2.56x10(9) and 1.33x10(9) genomic copies/ml respectively. This produced preliminary estimates of genomic copy/TU ratios of 34:1 and 18:1. However standard transduction conditions did not deplete fully the supernatant of transducing particles since the same supernatant was subsequently able to achieve 25% the initial transduction efficiency, although

... Determine retroviral transduction efficiency and gene expres...We describe three new ViraPort retroviral reporter vectors: pFB-hrGFP ...Dispensing with marker g...,Versatile,Reporter,Vectors,for,Monitoring,Viral,Transduction,biological,advanced biology technology,biology laboratory technology,biology device technology,latest biology technology

Retroviral (MLV-based) and lentiviral (HIV-1-based) vectors were harvested once at 1.5 days and again at 2.5 days post-transfection. Retroviral genomic particles pseudotyped with VSVGs (gMLV-VSVG) were produced at a higher titer during the first harvest than during the second harvest (Fig. 1A). However, more retroviral transducing particles (functional ones, tMLV-VSVG) were produced during the second harvest than during the first harvest. The number of gMLV-VSVG particles required for the transduction of a single cell (genomic to transducing particle ratio) can be calculated to be 2,200 for the first harvest, but 1,200 for the second harvest. This result indicates that gMLV-VSVG particles assembled at later time points post-transfection are more infectious than the ones assembled earlier.. In contrast, both lentiviral genomic and transducing particles pseudotyped with VSVGs (gHIV1-VSVG and tHIV1-VSVG, respectively) were produced at a higher titer during the first harvest than during the second ...

Background. Gene therapy with LentiGlobin Drug Product (DP), which contains autologous CD34+ hematopoietic stem cells transduced with the betibeglogene darolentivec (BB305) lentiviral vector, has been shown to eliminate red blood cell (RBC) transfusions in most patients with TDT with non-β0/β0 genotypes in the prior HGB-204 (Northstar) phase 1/2 study, with a safety profile consistent with myeloablative conditioning. A key finding in HGB-204 was that the average number of therapeutic gene copies per CD34+ cell (i.e. vector copy number [VCN] per diploid genome; median 0.7, range 0.3-1.5) in the DP correlated with peripheral HbAT87Q (transgenic hemoglobin [Hb]) expression at 6 months. To optimize the proportion of patients able to achieve transfusion independence and increase unsupported Hb levels after treatment, a refined manufacturing process for LentiGlobin DP was used, to increase the DP VCN and the proportion of transduced cells. Herein, we report initial data from the phase 3 HGB-207 ...

1 ? What is the name of the process of bacterial â ¦ Specialized transduction is instrumental in the isolation of the genes in molecular biology, and in the discovery of insertion elements, which often serve as attachment sites for phage DNA integration. An Introduction to Genetic Analysis. Based on how the DNA is packaged within the viral particle, there are two types of transduction: In generalized transduction, phage mistakenly packages bacterial DNA instead of their own phage DNA during phage assembly. Plasmid pQSR50, which contains transposon Tn 5 and encodes kanamycin and streptomycin resistance, was used in plasmid transduction assays. There are two kinds of transduction â specialized and generalized. Test your knowledge of the following with the quiz/worksheet combo: Be sure to check out the associated lesson, Bacterial Transduction: Definition, Process & Advantages, for further details. It is an example of vertical gene transfer. Answer: A. Save my name, email, and website in this ...

Confocal micrograph of NIH-3T3 cells co-transduced with 5 fluorescent proteins. The cells were marked by co-transduction with Lentiviral Gene Ontolog...

Inhibitory effect of adenovirus-uteroglobin transduction on the growth of lung cancer cell lines. - Jae Cheol Lee, Kyung-Ho Park, Seon Jin Han, Chul-Gyu Yoo, Choon-Taek Lee, Sung Koo Han, Young-Soo Shim, Young Whan Kim

Many are downloadable. The mechanism of specialized transduction is illustrated in Figure 4.Phages that mediate generalized transduction generally breakdown host DNA … If you want Generalized transduction vs Specialized Transduction NEET Video , EduRev notes & Videos, you can search for the same too. Meaning of Transduction: The transfer of a small part of a bacterial genome from a donor to recipient bacterium through the agency of a bacteriophage is called transduction. In Transduction, DNA is transferred from one cell to another through the agency of viruses. You can also find Generalized transduction vs Specialized Transduction NEET Video , EduRev ppt and other NEET slides as well. ADVERTISEMENTS: In this article we will discuss about the meaning and types of transduction of bacteria. Recombination is the major and most basic factor that increases and decreases chromosomal and … Enjoy the videos and music you love, upload original content, and share it all with friends, family, and the ...

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Our Human Fibroblast Immortalization Kits include an Immortalization Reagent and Virus Transduction Enhancer, increasing transduction rate up to 10-fold.

Figure 1. Promoter activity varies across several human and rodent cell lines. Cells were plated at a density of 50,000 cells per well in a 24-well plate and transduced at MOI = 15 with SMARTvector Empty Vector Control Particles expressing TurboGFP. Promoter activity was assessed at 72 hours post-transduction by the fluorescence intensity of TurboGFP.. If you are uncertain of relative promoter activity in your cells, the Dharmacon SMARTchoice Promoter Selection Plate and the SMARTchoice Inducible Non-targeting Control 4-pack allow straightforward identification of the optimal promoter in your cells of interest.. ...

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Do you know if lentivirus based vectors (allowing stable transduction of cells) insert to host DNA at specific sequences or randomly? I know it is a replication independent process ...

The Signal Transduction and Therapeutics Program Area is composed of 37 members, spanning 16 Departments within UCLA. In the past competing cycle, investigators...

The Cen-ters for Dis-ease Con-trol and Pre-ven-tion plans to scale back or dis-con-tinue work to pre-vent in-fec-tious-dis-ease epi-demics, other health threats in 39 for-eign coun-tries be-cause it ex-pects fund-ing to end, the agency told em-ploy-ees. https://t.co/ ...

The liver is an attractive organ for gene therapy because of its important role in many inherited and acquired diseases. Recombinant adeno-associated viruses (rAAVs) have been shown to be good candidates for liver gene delivery, leading to long-term gene expression. We evaluated the influence of the route of administration on rAAV-mediated liver transduction by comparing levels of luciferase expression in the livers of male and female mice after injection of rAAV serotype 2, using three different routes of administration: intravenous (IV), intraportal (IP), or direct intrahepatic (IH) injection. To determine transgene expression we used a noninvasive optical bioluminescence imaging system that allowed long-term in vivo analysis. After IV injection dramatic differences in liver transgene expression were observed, depending on gender. When IP injection was used the differences were reduced although they were still significant. Interestingly, direct intrahepatic injection of rAAV vectors was ...

The present study was undertaken to analyse the capability of HIV-1 derived TAT protein transduction domain (PTD) fused with Green Fluorescent Protein (TAT-GFP) as a delivery vehicle into a range of protozoan parasites. Successful transduction of native purified TAT-GFP was observed by fluorescent microscopy in Cryptosporidium parvum, Giardia duodenalis, and Neospora caninum. The ability to transduce peptides and other cargo into protozoan parasites, will greatly assist in the delivery of future peptide-based drugs and target validation peptides.. ...

The effect of chemotherapy drug Mitomycin C (MMC) in combination with recombinant adeno-associated virus II (rAAV2) in cancer therapy was investigated, and the mechanism of MMC affecting rAAV2s bioactivity was also studied. The combination effect was evaluated by the level of GFP and TNF expression in a human glioma cell line, and the mechanism of MMC effects on rAAV mediated gene expression was investigated by AAV transduction related signal molecules. C57 and BALB/c nude mice were injected with rAAV-EGFP or rAAV-TNF alone, or mixed with MMC, to evaluate the effect of MMC on AAV-mediated gene expression and tumor suppression. MMC was shown to improve the infection activity of rAAV2 both in vitro and in vivo. Enhancement was found to be independent of initial rAAV2 receptor binding stage or subsequent second-strand synthesis of target DNA, but was related to cell cycle retardation followed by blocked genome degradation. In vivo injection of MMC combined with rAAV2 into the tumors of the animals

PubMed journal article: [Development and application of a safe SARS-CoV neutralization assay based on lentiviral vectors pseudotyped with SARS-CoV spike protein]. Download Prime PubMed App to iPhone, iPad, or Android

Lenti-X Accelerator is a magnetic bead-based technology designed to accelerate lentiviral transduction and MMLV retrovirus transduction experiments. Lenti-X Accelerator allows you to complete this process in only 30 minutes, without Polybrene.

Lenti-X Accelerator is a magnetic bead-based technology designed to accelerate lentiviral transduction and MMLV retrovirus transduction experiments. Lenti-X Accelerator allows you to complete this process in only 30 minutes, without Polybrene.

A recombinant adeno-associated virus serotype 2 (rAAV2) vector has been altered to carry the human MERTK (hMERTK) gene. This vector has been shown to restore vision in animal models that resemble human MERTK-associated Retinitis Pigmentosa (RP), an incurable retinal degeneration that causes severe vision loss. The proposed study is an open label, Phase I clinical trial of subretinal rAAV2-VMD2-hMERTK administration to individuals with MERTK-associated retinal disease. This trial will lead to a greater understanding of the safety and thereby potential value of gene transfer in MERTK-associated retinal disease and will have implications for other forms of retinal degenerative disease amenable to this type of intervention ...

A recombinant adeno-associated virus serotype 2 (rAAV2) vector has been altered to carry the human MERTK (hMERTK) gene. This vector has been shown to restore vision in animal models that resemble human MERTK-associated Retinitis Pigmentosa (RP), an incurable retinal degeneration that causes severe vision loss. The proposed study is an open label, Phase I clinical trial of subretinal rAAV2-VMD2-hMERTK administration to individuals with MERTK-associated retinal disease. This trial will lead to a greater understanding of the safety and thereby potential value of gene transfer in MERTK-associated retinal disease and will have implications for other forms of retinal degenerative disease amenable to this type of intervention ...

Recombinant Adeno-associated virus 2 ATCC ® VR-1616™ Designation: Recombinant Adeno-associated Virus 2 Reference Standard Stock (rAAV2 RSS) Application:

Recombinant Adeno-associated virus 2 ATCC ® VR-1616™ Designation: Recombinant Adeno-associated Virus 2 Reference Standard Stock (rAAV2 RSS) Application:

TY - JOUR. T1 - G2 Cell Cycle Arrest and Cyclophilin A in Lentiviral Gene Transfer. AU - Zhang, Shangming. AU - Joseph, Guiandre. AU - Pollok, Karen. AU - Berthoux, Lionel. AU - Sastry, Lakshmi. AU - Luban, Jeremy. AU - Cornetta, Kenneth. PY - 2006/10. Y1 - 2006/10. N2 - Lentiviral vectors derived from the human immunodeficiency virus-1 (HIV-1) have a higher propensity to transduce nondividing cells compared to vectors based on oncoretroviruses. We report here that genistein, a previously known protein tyrosine kinase (PTK) inhibitor and G2 cell cycle arrest inducer, significantly enhanced lentiviral transduction in a dose-dependent manner. Increased transduction, as measured by vector expression, was seen in a variety of human cell lines, murine primary lymphocytes, and primary human CD34+ peripheral blood progenitor cells as well. Increased vector expression was also associated with an increase in vector DNA copy number, as assessed by quantitative PCR. Genistein-mediated G2 cell cycle arrest, ...

Because the features and kinetics of adeno−associated virus(AAV)−mediated gene transfer to endothelial cells(EC)are yet to be ultimately determined, we tested variables pertinent to the efficiency of AAV−mediated gene transfer to bovine aortic endothelial cells(BAEC).The variables with AAV vectors were compared with the better characterized adenovirus(Ad)vectors.There is a dose−response relationship between multiplicity of infection(moi)of AAV or Ad vectors and transduction efficiency in BAEC.The higher moi of AAV vectors achieved more than 80% of transduction efficiency in cultured BAEC.AAV and Ad vectors showed an incubation−time−dependent increase in transduction efficiency of LacZ gene to the BAEC up to 12 h of vector exposure.Although the similar kinetics of transduction efficiency of LacZ gene to BAEC was found in both vectors, the duration of gene expression was longer in AAV vector than that in Ad vectors in vitro.These results indicate that AAV−vector is efficient for gene ...

Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related dependovirus AAV-3. We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2. The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese

Recombinant vectors based on adeno-associated virus (rAAV) are promising tools to specifically alter complex genomes through homologous recombination (HR)-based gene targeting. In a therapeutic setting, an AAV donor vector will recombine with a mutant target locus in order to correct the mutation di …

Background- Viral delivery vectors founded on the adeno-associated virus (AAV) serotype 2 platform are currently being evaluated for human gene transfer. All viral genes can be removed, resulting in only the transgene of interest and the viral terminal repeats. These small non-enveloped single-stranded DNA viruses have been associated with long duration expression in experimental animals, and these qualities make AAV serotypes ideal systems when considering viral gene delivery. However, while scores of serotypes have been described, few have been compared directly. In this study we have directly compared AAV serotypes 1-9 by two routes of injection, skeletal muscle and tail vein.. Methods and Results- Previous studies of AAV serotypes 1-5 have demonstrated uniformity in production and purification. Those results guided this comparison of luciferase gene expression-efficiency between AAV serotypes 1-9 after single injection into mouse muscle, or tail vein, of 1 x1011 viral particles into the ...

PURPOSE: Treatment of inherited retinal degenerations using adeno-associated viral (AAV) vectors involves delivery by subretinal injection. In the latter stages, alteration of normal anatomy may cause difficulty in visualizing the retinotomy, retinal detachment extension, and vector diffusion. Vital dyes may be useful surgical adjuncts, but their safety and impact on AAV transduction are largely unknown. METHODS: The effects of Sodium Fluorescein (SF), Membrane Blue (MB), and Membrane Blue Dual (DB) at a range of dilutions were assessed on human embryonic kidney cells in vitro using an AAV2-green fluorescent protein (GFP) reporter at different multiplicities of infection. Flow cytometry analysis was performed to assess both cell viability and transduction efficiency. The effect on quantitative (q)PCR titer was determined. Balanced salt solution (BSS) or dilute DB (1:5 in BSS) were delivered subretinally into left/right eyes of C57BL/6J mice (n = 12). Retinal structure and function were analyzed by

The development of lentiviral vectors to deliver genes to specific cell types are useful tools because they have the ability to produce stable transduction, maintain long-term transgene expression, and transduce both dividing and non-dividing cells. Despite the high transduction efficiency of lentiviral vectors, their tropism frequently does not match the desired gene delivery application. We report herein, strategies to modify lentiviral vectors using diverse techniques which allow targeting gene delivery to specific cell types. To target CD117 expressing cells we engineered a lentivector that incorporates membrane-bound human stem cell factor (hSCF), and for fusion, a Sindbis virus-derived fusogenic molecule (FM) onto the lentiviral surface. Lentiviral vectors pseudotyped with envelope proteins of alphaviruses have recently attracted considerable interest for their potential utilization for immunotherapy due to their capacity to transduce dendritic cells. We report lentiviral vectors ...

Combined expression of costimulatory factors and proinflammatory cytokines stimulate effective immune-mediated tumor rejection in a variety of murine tumor models. Specifically, syngeneic tumor cells genetically modified to express B7.1 (CD80) have been shown to induce rejection of previously established murine solid tumors, and transduction with IL-2 can further increase survival. However, poor rates of gene transfer and inefficient expression of multiple transgenes encoded by single vectors have hampered the development of such autologous tumor cell vaccines for clinical trials in acute myeloid leukemia (AML) patients. Here we describe the development of a self-inactivating lentiviral vector encoding B7.1 and IL-2 as a single fusion protein postsynthetically cleaved to generate biologically active membrane-anchored B7.1 and secreted IL-2. This enables the efficient transduction of both established and primary AML blasts, resulting in expression of the transgenes in up to 98% of the cells following a

Gene transfer to the gastrointestinal (GI) mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD), GI infections, and cancer. We evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G)-pseudotyped lentiviral vectors (LV) for efficacy of gene transfer to both murine rectosigmoid colon in vivo and human colon explants ex vivo. LV encoding beta-galactosidase (LV-β-Gal) or firefly-luciferase (LV-fLuc) reporter genes were administered by intrarectal instillation in mice, or applied topically for ex vivo transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by ex vivo bioluminescence imaging. LV-β-Gal expression

Background. Patients with severe sickle cell disease (SCD) may benefit from β-globin gene transfer into autologous hematopoietic stem cells (HSC). Successful HBB gene transfer requires vector-mediated transduction of primitive HSCs. Steady-state bone marrow (BM) is the default HSC source in patients with SCD. Normal human BM contains up to 30% CD34+CD19+ pro-B cells and other lineage-committed cell types (CD34dim) that will not contribute to improved long-term erythropoiesis via gene therapy; these cells mobilize at low rates. CD34+ cell yields from BM harvest (BMH) are typically lower than those after mobilization and peripheral blood (PB) apheresis; multiple rounds of BMH may be required to obtain adequate cell doses for autologous gene therapy (GT) protocols. As G-CSF can cause life-threatening SCD complications and is contraindicated, plerixafor, a CXCR4 receptor antagonist, may accomplish HSC mobilization without the neutrophil or endothelial activation that elicit vaso-occlusion. We ...

Protein transduction domains (PTDs), both naturally occurring and synthetic, have been extensively utilized for intracellular delivery of biologically active molecules both in vitro and in vivo. However, most comparisons of transduction efficiency have been performed using fluorescent markers. To compare efficiency of functional protein transduction, a peptide derived from IkB kinase ß (IKKß) that prevents formation of an active IKK complex was used as a biologically active cargo. This peptide, termed NEMO Binding Domain (NBD), is able to block activation of the transcriptional factor NF-κB by IKK, but not basal NF-κB activity. Our results demonstrate that Antp and Tat PTDs were most effective for delivery of NBD for inhibition of NF-kB activation compared to other PTD-NBD in both Hela and 293 cells, however, at higher concentrations (100 µM), the Antp-NBD as well as the FGF-NBD peptide caused significant cellular toxicity. In contrast to the cell culture results, delivery of NBD using 8K

Cornerstone for an efficient cardiac gene therapy is the need for a vector system which enables selective and long-term expression of the gene of interest. In rodent animal models pseudotyped adeno-associated viral vectors like AAV2/6 have been shown to be superior in the transduction of cardiomyocytes compared to other AAV serotypes. However, since significant species dependent differences in transduction characteristics exist, and large animal models are of imminent need for pre-clinical evaluations, we compared the gene transfer efficiency of an AAV2/6 and a heparin-binding site deleted AAV2 (AAV2/dHep) in a porcine model.. Methods: Viral vectors were produced using a pseudotyping strategy. All vectors did express the reporter gene luciferase under control of a cardiac specific promoter (MLC2v). Vectors were delivered via percutaneous pressure-regulated retroinfusion into the coronary vein (3.5 x 10e10 genome copies/animal; n = 5 animals/group). Expression levels were evaluated 4 weeks after ...

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title: Analysis of the production efficiency and titration of various recombinant adeno-associated viruses, doi: none, category: Article

Adenovirus serotype 5 (Ad5) is widely used as an oncolytic agent for cancer therapy. However, its infectivity is highly dependent on the expression level of coxsackievirus-adenovirus receptor (CAR) on the surface of tumor cells. We engineered Ad5 virus with the protein transduction domain (PTD) from the HIV-1 Tat protein (Tat-PTD) inserted in the hypervariable region 5 (HVR5) of the hexon protein in the virus capsid. Tat-PTD-modified Ad5 shows a dramatically increased transduction level of CAR-negative cells and bypassed fiber-mediated transduction. It also overcomes the fiber-masking problem, which is caused by release of excess fiber proteins from infected cells. To achieve specific viral replication in neuroblastoma and neuroendocrine tumor cells, we identified the secretogranin III (SCG3) promoter and constructed an adenovirus Ad5PTD(ASH1-SCG3-E1A) wherein E1A gene expression is controlled by the SCG3 promoter and the achaete-scute complex homolog 1 (ASH1) enhancer. This virus shows ...

Lentiviral vectors have emerged as efficient tools for investigating T cell biology through their ability to efficiently deliver transgene expression into both dividing and nondividing cells. Such lentiviral vectors have the potential to infect a wide variety of cell types. However, despite this advantage, the ability to transduce primary human T cells remains challenging and methods to achieve efficient gene transfer are often time consuming and expensive. We describe a method for generating lentivirus that is simple to perform and does not require the purchase of non-standard equipment to transduce primary human T cells. Therefore, we provide an optimized protocol that is easy to implement and allow transduction with high efficiency and reproducibility.

Lentiviral vectors are tools for gene transfer derived from lentiviruses. From their first application to now they have been strongly developed in design, in biosafety and in their ability of transgene expression into target cells. Primate and non-primate derived lentiviral vectors are now available …

Adeno-associated virus (AAV) vectors are a common platform for in vivo gene therapy applications and have been used successfully in clinical trials in patients with blood diseases and blindness. The presence of pre-existing neutralizing antibodies in about 50% of patients significantly decreased the clinical utility of AAV vectors. The prevailing assays used to screen for AAV neutralizing antibodies are not standardized, and can produce unreliable data depending on the AAV serotype. Drs. George Aslanidi and Karina Krotova at the University of Minnesota have developed a fast and reliable method to evaluate pre-existing AAV neutralizing antibodies for most of the commonly used AAV serotypes. The resulting assay can be used with virtually all AAV serotypes, requires small amounts of AAV and can produce reliable data in 24 hours. ...

Adeno-associated virus (AAV) is a member of the family parvoviridae that has been widely used as a vector for gene therapy because of its safety profile, its ability to transduce both dividing and non-dividing cells, and its low immunogenicity. AAV has been detected in many different tissues of several animal species but has not been associated with any disease. As a result of natural infections, antibodies to AAV can be found in many animals including humans. It has been shown that pre-existing AAV antibodies can modulate the safety and efficacy of AAV vector-mediated gene therapy by blocking vector transduction or by redirecting distribution of AAV vectors to tissues other than the target organ. This review will summarize antibody responses against natural AAV infections, as well as AAV gene therapy vectors and their impact in the clinical development of AAV vectors for gene therapy. We will also review and discuss the various methods used for AAV antibody detection and strategies to overcome

Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics. Here we describe a capsid selection method, called Cre recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV9 (refs. 14,15,16,17), and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to ...

Amato R, Morleo M, Giaquinto L, di Bernardo D, Franco B (2014). A network-based approach to dissect the cilia/centrosome complex interactome. BMC Genomics 15 658.. Ayuso E, Blouin V, Lock M, McGorray S, Leon X, Alvira MR, Auricchio A, Bucher S, Chtarto A, Clark KR, Darmon C, Doria M, Fountain W, Gao G, Gao K, Giacca M, Kleinschmidt J, Leuchs B, Melas C, Mizukami H, Muller M, Noordman Y, Bockstael O, Ozawa K, Pythoud C, Sumaroka M, Surosky R, Tenenbaum L, van der Linden I, Weins B, Wright JF, Zhang X, Zentilin L, Bosch F, Snyder RO, Moullier P (2014). Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material. Hum Gene Ther 25 (11):977-987.. Belcastro V, di Bernardo D (2014). Reverse engineering transcriptional gene networks. Methods Mol Biol 1101 179-196.. Bello A, Chand A, Aviles J, Soule G, Auricchio A, Kobinger GP (2014). Novel adeno-associated viruses derived from pig tissues transduce most major organs in mice. Sci Rep 4 6644.. Beznoussenko ...

Binbin Cheng is the author of this article in the Journal of Visualized Experiments: High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Cell culture and transient transfection. Mouse WT and Cd2ap-/- (18) podocytes were cultured as described previously (42). HEK 293 cells were maintained and transfected using Lipofectamine 2000 reagent (Invitrogen) as previously reported (6). Adenoviral infections of cultured podocytes were preformed as described previously (6). Lentiviral infection. Lentiviral shRNA plasmids for CatL were obtained from Open Biosystems and were used to generate lentiviral transduction particles in HEK 293T cells. We used a target set of 3 clones with pLKO.1,-puro as the parental vector (see Supplemental Table 1 for sequences). Lentiviral shRNA plasmids for dendrin were obtained from Sigma Aldrich and were used to generate lentiviral transduction particles in HEK 293T cells. We used a target set of 2 clones with pLKO.1,-puro as a parental vector (Supplemental Table 1). Lentiviral knockdown of mouse CatL and dendrin were performed in differentiated mouse and high-Tgfb1Cd2ap-/- podocytes according to the protocol ...

BACKGROUND AND OBJECTIVE: Gene transfer and expression of exogenous genetic information coding for an immunogenic protein in antigen presenting cells (APCs) can promote an immune response. This was investigated by retroviral transfer of a marker gene into CD34+ derived APCs. DESIGN AND METHODS: To achieve long term expression of a specific transgene in APCs, G-CSF mobilized peripheral blood CD34+ cell populations were retrovirally transduced with the bacterial nlsLacZ, a marker gene used here as a model, in the presence of IL-3, IL-6, GM-CSF and SCF prior to being induced to differentiate into dendritic and macrophage cells by GM-CSF and TNF-a. RESULTS: Addition of IL-4 was found to induce dendritic differentiation preferentially by inhibiting proliferation and differentiation of the macrophage lineage. As assessed by X-Gal staining, LacZ gene expression was observed in cells from both the dendritic lineage (CD1a+/CD14-) which still exhibits the highest immunostimulatory activity in mixed ...

295395284 - EP 1032678 A1 2000-09-06 - HORMONE-DEPENDENT FORMS OF THE ADENO-ASSOCIATED VIRUS, REP PROTEINS, DNA SEQUENCES CODING FOR THEM, VECTORS CONTAINING THEM, AND REGULATORY METHODS OF THEIR INTRACELLULAR ACTIVITY - [origin: WO9927110A1] The subject matters of this invention are the hormone-dependent forms of Rep 78 and Rep 68 proteins of the Adeno-associated virus (AAV), obtained by the fusion of their specific mutants with the hormone binding domain (HBD) of steroid hormone receptors, and the DNA sequences coding for them. The invention also refers to a method for the hormonal regulation of the activity of the fusion products Rep78/68-HBD, inserted into eucaryotic cells utilising viral or non-viral systems, in order to direct the stable integration of DNA sequences in specific regions of the host human genome for therapeutic purposes. The fusion products Rep78/68-HBD are also utilised to generate viral hybrid vectors (i.e. adenovirus vectors AAV) and for generating recombinant vectors AAV.[origin

The use of chimeric antigen receptor (CAR)-redirected CD8+ T cells in the treatment of malignancy is a novel strategy that has shown tremendous promise; however, it remains likely this strategy could be enhanced when combined with additional approaches. We hypothesized that deletion of signaling mediators that negatively regulate TCR signal transduction might enhance CAR signaling and function, since CARs utilize the same machinery that transduces TCR signals. We tested CAR activity and function in T cells that lacked one or both lymphocyte-specific isoforms of diacylglycerol kinase (dgk), proteins that metabolize the second messenger DAG and limit Ras/ERK activation. We found that primary murine T cells transduced with CARs specific for the human tumor antigen mesothelin demonstrated greatly enhanced cytokine production and cytotoxicity when co-cultured with a murine mesothelioma line that stably expresses mesothelin. Additionally, we found that dgk-deficient CAR transduced T cells were more ...

OBJECTIVE: Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease gene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single inverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here we aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems. METHODS: Oversized (|8kb) transgene constructs containingABCA4coding sequence were packaged as truncated fragments |5kb in size into various AAV serotypes.In vitrotransductions with these fAAV vector preparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and western blot techniques. RESULTS: Transductions with fAAV vector preparations yieldedABCA4mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full lengthABCA4CDS with additional

TY - JOUR. T1 - Interaction between endothelial cells and the secreted cytokine drives the fate of an IL4- or an IL5-transduced tumour. AU - Di Carlo, Emma. AU - Modesti, Andrea. AU - Coletti, Anna. AU - Colombo, Mario P.. AU - Giovarelli, Mirella. AU - Forni, Guido. AU - Diodoro, Maria G.. AU - Musiani, Piero. PY - 1998. Y1 - 1998. N2 - Injection of interleukin-4 (IL4) gene-transduced tumour cells into syngeneic immunocompetent mice resulted in turnout rejection in which a key role for eosinophils was suggested. To evaluate whether IL5 inhibits turnout growth by selectively inducing eosinophil recruitment and activation, a poorly differentiated mammary adenocarcinoma cell line (TSA) was transfected with the IL5 gene and the cells secreting IL5 (TSA-IL5) were injected subcutaneously (s.c,) in syngeneic mice. The oncogenicity of TSA-IL5 was compared with that exhibited by TSA cells transfected with the IL4 gene (TSA- IL4) and with the neomycin resistance gene only (TSA-neo). At progressive times ...

This study is aiming to determine whether administration of tretinoin, a myeloid-derived suppressor cell inhibitor, with a wild-type p53-transduced cancer

The acquired immunodeficiency syndrome (AIDS) was first described about 20 years ago. Since then almost 20 million people have died from human immunodeficiency virus (HIV-1) infection and 42 million are infected with. New drugs and an effective vaccine are urgently needed. In particular, new drugs that block the HIV type 1 (HIV-1) entry into host cell have clear advantages over the currently used drugs. They should abrogate the establishment of a productive infection and consequently could diminish the chances of HIV-1 developing resistance. Furthermore, a vaccine that prevents AIDS should elicit broadly cross-reactive neutralizing antibodies to prevent infection. A safe and simple assay for measuring neutralizing activities against different HIV-1 strains is critical for the development of such a vaccine or entry inhibiting drugs.. We previously generated a retroviral vector which specifically transfers genes into human CD4+ cells [1, 2]. This vector was derived by pseudotyping murine leukemia ...

Monitor signal transduction in real time. With SBIs line of pGreenFire1 Pathway Reporters, you can monitor signal transduction in real time. These vectors leverage our reliable lentivector technology and save you time-our pre-built signal transduction pathway reporters come as ready-to-package lentivector plasmid and ready-to-transduce pre-packaged lentivirus*. The pGreenFire1-MEF2 (EF1α-neo) Lentivector co-expresses a destabilized copepod GFP (dscGFP; 2-hour half-life) and luciferase from MEF2 transcriptional response elements (TREs) paired with a minimal CMV promoter (mCMV). The mCMV promoter alone delivers negligible expression, but when downstream of MEF2-responsive transcriptional elements, drives expression of dscGFP and luciferase in response to MEF2 activity. The result is the ability to quantitatively measure MEF2 activity by fluorescence and luciferase activity ...

SBIs lentivector-based imaging systems offer safe, stable, and effective gene delivery in virtually all mammalian cells in tissue culture and in whole animal models.. The CMV-Luciferase-RFP-TK Lentivector for In Vivo Imaging leverages SBIs well-regarded lentivector technology for either transduction or transfection of the reporter into target cells. Because the vector becomes integrated into the genome, you can use this vector to create stable reporter cell lines.. The vector expresses RFP, luciferase, and TK from a CMV promoter for strong expression in most common cell types, such as HeLa, HEK293, or HT1080. Available as either a plasmid or pre-packaged virus.. Which BLIV construct should you choose?. With a range of options, SBIs vectors for in vivo imaging support a wide range of projects. Simply choose the vector that best fits your needs:. Choose a lentivector when:. ...

The second question a researcher might ask is: What type of cells do I want to control, and how I can express the opsin specifically in that cell type? Gene coding for light-sensitive proteins can be delivered to target cells by transfection, viral transduction, or the creation of transgenic animal lines. Expression can be restricted to cells of interest using specific promoters or recombinase-based conditional systems. The use of viral vectors for gene delivery can allow targeting of specific cells without specific promoters, for example, to target neuronal populations based on their topological connections. Lenti and adeno-associated viral vectors have been used successfully to introduce opsins into the mouse, rat, and primate brain. Additionally, these have been well tolerated and highly expressed over long periods of time, with no reported adverse effects. However, a major downside of viral expression systems is the maximum genetic payload length; only promoter fragments that are small (less ...

This book is great to learn, produce, and apply several different types of viral and non-viral vectors for gene transfer. It provides all necessary information from beginning to the end for successful genaration of vectors. Troubleshooting guidelines is also very helpful ...

It has been previously shown that TCR-transduced bone marrow cells controlled the growth of human tumors in severe combined immunodeficiency mice (15). It has also been shown that TCR transduction of HSCs could mediate antitumor immunity (16, 17). However, the approach to obtain a number of HSCs or ESCs from cancer patients is often not feasible. Recent iPS cell technology can generate iPS cells from patients without any surgical approach. Thus, iPS cells have greater potential to be used in ACT-based therapies. Our study significantly facilitates this application.. Although TCR-transduced iPS cells need up to 6 to 8 weeks to develop into fully differentiated T cells, there are possibilities to enhance this development. Researchers have evaluated the efficacy of ACT therapy by transferring tumor-specific CD8+ T cells at various stages of differentiation into tumor-bearing mice. These studies concluded that administration of naïve and early effector T cells, in combination with a lymphodepleting ...

Accumulating evidence implicates monopolar spindle-one-binder protein (MOB)2 as an inhibitor of nuclear-Dbf2-related kinase (NDR) by competing with MOB1 for interaction with NDR1/2. vector-transduced cells. By contrast, the overexpression of MOB2 resulted in the opposite results. Mechanistically, MOB2 controlled the Anamorelin distributor alternative connection of MOB1 with NDR1/2 and LATS1, which led to elevated phosphorylation of LATS1 and MOB1 and thus resulted in the inactivation of YAP and therefore inhibition of cell motility. The outcomes of todays study provide proof MOB2 serving an optimistic function in LATS/YAP activation by activating the Hippo Anamorelin distributor signaling pathway. solid course=kwd-title Keywords: monopolar spindle-one-binder protein 2, hippo pathway, yes-associated protein, nuclear-Dbf2-related kinase, large tumor suppressor, monopolar spindle-one-binder protein 2 Introduction Monopolar spindle-one-binder proteins (MOBs) are highly conserved from yeast to ...

Retroviral replicating vectors (RRVs) has been proved as an excellent gene therapy vector to achieve efficient gene transduction.

VIVEbiotech is a cdmo specialized in the development and manufacture of research, preclinical toxicology and gmp-grade lentiviral vectors.

Supplementary MaterialsS1 Table: Pre-existing prevalence of NAb against numerous AAV serotypes in local felines shown for every canton of Switzerland. and without NAb against each AAV serotype or all Imiquimod inhibitor mixed had been analyzed using the MannCWhitney U check (ptransduction by 50%.(PDF) pone.0212811.s006.pdf (1.9M) GUID:?6DEA2Stomach1-E3DE-461D-8BA1-EF6C3F611C0B S4 Fig: Existence of NAb against AAV-DJ in preferred client-owned domestic felines. A transduction inhibition assay was performed to be able to determine Imiquimod inhibitor the current presence of NAb against AAV-DJ-EGFP. Twenty kitty serum examples proven to have NAb against various other AAV serotypes were particular previously. Two different serum dilutions had been tested for every test: 1:10 and Imiquimod inhibitor 1:20. Handles included an AAV-NAb-positive individual serum (H2 1:20), a non-serum control (AAV-DJ) and cells just (mock-infection with out a trojan; Neg.). Explanations within each picture make reference ...

Speedy and delicate detection of interleukin-6 in serum through time-resolved lateral movement immunoassay. Interleukin 6 (IL-6) is an interleukin that acts as each a proinflammatory and anti inflammatory cytokine. It may be used as a possible diagnostic biomarker for sepsis. The intention of this research was to determine an easy-to-use detection equipment for speedy, quantitative and on-site … ...

Recombinant virus particles synthesized within the packaging cell lines lack the Gag, Pol and Env genes. Therefore, these viruses are said to be replication defective; in other words, they are rendered incapable of producing additional viruses. Such viruses are considered ideal for transfecting cells within a host to facilitate the targeted delivery of therapeutic genes.

Speedy and delicate detection of interleukin-6 in serum through time-resolved lateral movement immunoassay. Interleukin 6 (IL-6) is an interleukin that acts as each a proinflammatory and anti inflammatory cytokine. It may be used as a possible diagnostic biomarker for sepsis. The intention of this research was to determine an easy-to-use detection equipment for speedy, quantitative and on-site … ...

Signal transduction is any process by which a cell converts one kind of signal or stimulus into another. Processes referred to as signal transduction often involve a sequence of biochemical reactions inside the cell, which are carried out by enzymes and linked through second messengers. In many transduction processes, an increasing number of enzymes and other molecules become engaged in the events that proceed from the initial stimulus. Responses of cells to environmental signals, toxins and stressors have profound implications for diverse aspects of human health and disease including development, cystic fibrosis, diabetes, asthma, heart, autoimmune diseases and cancer. The delineation of the signal transduction pathways affected in these and other complex human diseases are likely to present new avenues for therapeutic intervention and understanding of human disease mechanisms ...

Methodology provided: The Conrad group will provide expertise for the development of new in vivo models for the study of important players in thiol dependent processes. Concerning mouse analyses, the group has easy access to the German Mouse Clinic (headed by Prof. M. Hrabé de Angelis) for phenotypical and behavioral studies of transgenic mouse models. Furthermore, in vitro investigations of cell lines established from mutant mice along with highly efficient lentiviral add-back systems for the gene of interest are essential tools to study cellular and biochemical mechanisms. In addition, the group has established cell-based screening platforms for large scale drug discovery approaches (in collaboration with Dr. Joel Schick ...