The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events

Background: Although lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events. Methods: Lentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector - target cell contact and adsorption periods were studied. MOI between 0-32 was assessed on commonly used cell lines as well as a new cell line. Results: We demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0-32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained. Conclusion: Several variables in the transduction process affected in in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted. © 2004 Zhang et al; licensee BioMed Central Ltd.

The effect of transforming growth factor β1 gene polymorphisms in ankylosing spondylitis

Objectives. To determine whether genetic polymorphisms in or near the transforming growth factor β1 (TGFB1) locus were associated d with susceptibility to or severity of ankylosing spondylitis (AS). Methods. Five intragenic single-nucleotide polymorphisms (SNP) and three microsatellite markers flanking the TGFB1 locus were genotyped. Seven hundred and sixty-two individuals from 184 multiplex families were genotyped for the microsatellite markers and two of the promoter SNPs. One thousand and two individuals from 212 English and 170 Finnish families with AS were genotyped for all five intragenic SNPs. A structured questionnaire was used to assess the age of symptom onset, disease duration and disease severity scores, including the BASDAI (Bath Ankylosing Spondylitis Disease Activity Index) and BASFI (Bath Ankylosing Spondylitis Functional Index). Results. A weak association was noted between the rare TGFB1 + 1632 T allele and AS in the Finnish population (P = 0.04) and in the combined data set (P = 0.03). No association was noted between any other SNPs or SNP haplotype and AS, even among those families with positive non-parametric linkage scores. The TGFB1 +1632 polymorphism was also associated with a younger age of symptom onset (English population, allele 2 associated with age of onset greater by 4.2 yr, P = 0.05; combined data set, allele 2 associated with age of onset greater by 3.2 yr, P = 0.02). A haplotype of coding region SNPs (TGFB1 +869/ +915+1632 alleles 2/1/2) was associated with age of symptom onset in both the English parent-case trios and the combined data set (English data set, haplotype 2/1/2 associated with age of onset greater by 4.9 yr, P = 0.03; combined data set, haplotype 2/1/2 associated with greater age of onset by 4.2 yr, P = 0.006). Weak linkage with AS susceptibility was noted and the peak LOD score was 1.3 at distance 2 cM centromeric to the TGFB1 gene. No other linkage or association was found between quantitative traits and the markers. Conclusion. This study suggests that the polymorphisms within the TGFB1 gene play at most a small role in AS and that other genes encoded on chromosome 19 are involved in susceptibility to the disease.

Polymorphism in codon 17 of the CTLA-4 gene (+49 A/G) is not associated with susceptibility to rheumatoid arthritis in British Caucasians

The role of the CTLA-4 antigen in the development of autoimmune diseases is well documented, with several autoimmune disorders showing association or linkage with the CTLA-4 locus. Its role in the aetiology of rheumatoid arthritis (RA) however, remains unclear, as the functional studies of the B7-CTLA-4 pathway in mouse models of RA and genetic studies in humans have given contrasting results. We have studied the single nucleotide polymorphism at position +49 (A/G) of the CTLA-4 gene, in a cohort of 421 RA cases and 452 healthy controls from the UK. Despite the high statistical power to detect even a weak susceptibility effect, no significant association was found. We also analysed the distribution of the allele and genotype frequencies with respect to the presence of the shared epitope (a known RA susceptibility factor) and found no statistically significant differences. We conclude that, although the importance of the B7-CTLA-4 interaction in the development of RA can not be excluded, the CTLA-4 gene is unlikely to be a predisposing factor to this disease.

Polymorphisms of the CYP2D6 gene increase susceptibility to ankylosing spondylitis

Ankylosing spondylitis (AS) is a common and highly familial rheumatic disorder. The sibling recurrence risk ratio for the disease is 63 and heritability assessed in twins > 90%. Although MHC genes, including HLA-B27, contribute only 20-50% of the genetic risk for the disease, no non-MHC gene has yet been convincingly demonstrated to influence either susceptibility to the disease or its phenotypic expression. Previous linkage and association studies have suggested the presence of a susceptibility gene for AS close to, or within, the cytochrome P450 2D6 gene (CYP2D6, debrisoquine hydroxylase) located at chromosome 22q13.1. We performed a linkage study of chromosome 22 in 200 families with AS affected sibling-pairs. Association of alleles of the CYP2D6 gene was examined by both case-control and within-family means. For case-control studies, 617 unrelated individuals with AS (361 probands from sibling-pair and parent-case trio families and 256 unrelated non-familial sporadic cases) and 402 healthy ethnically matched controls were employed. For within-family association studies, 361 families including 161 parent-case trios and 200 affected sibling-pair families were employed. Homozygosity for poor metabolizer alleles was found to be associated with AS. Heterozygosity for the most frequent poor metabolizer allele (CYP2D6*4) was not associated with increased susceptibility to AS. Significant within-family association of CYP2D6*4 alleles and AS was demonstrated. Weak linkage was also demonstrated between CYP2D6 and AS. We postulate that altered metabolism of a natural toxin or antigen by the CYP2D6 gene may increase susceptibility to AS.

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed as CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate tumor cells were mixed with mouse blood cells and the labelfree isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.

Analysis of differential gene expression based on Bayesian estimation of variance

Gene expression is arguably the most important indicator of biological function. Thus identifying differentially expressed genes is one of the main aims of high throughout studies that use microarray and RNAseq platforms to study deregulated cellular pathways. There are many tools for analysing differentia gene expression from transciptomic datasets. The major challenge of this topic is to estimate gene expression variance due to the high amount of ‘background noise’ that is generated from biological equipment and the lack of biological replicates. Bayesian inference has been widely used in the bioinformatics field. In this work, we reveal that the prior knowledge employed in the Bayesian framework also helps to improve the accuracy of differential gene expression analysis when using a small number of replicates. We have developed a differential analysis tool that uses Bayesian estimation of the variance of gene expression for use with small numbers of biological replicates. Our method is more consistent when compared to the widely used cyber-t tool that successfully introduced the Bayesian framework to differential analysis. We also provide a user-friendly web based Graphic User Interface for biologists to use with microarray and RNAseq data. Bayesian inference can compensate for the instability of variance caused when using a small number of biological replicates by using pseudo replicates as prior knowledge. We also show that our new strategy to select pseudo replicates will improve the performance of the analysis. - See more at: http://www.eurekaselect.com/node/138761/article#sthash.VeK9xl5k.dpuf

The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication

Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.

Role of TATATAA element in the regulation of tRNA(1)(Gly) gene expression in Bombyx mori is position dependent

Transcription of tRNA genes by RNA polymerase III is controlled by the internal conserved sequences within the coding region and the immediate upstream flanking sequences. A highly transcribed copy of glycyl tRNA gene tRNA1(Gly)-1 from Bombyx mori is down regulated by sequences located much farther upstream in the region -150 to -300 nucleotides (nt), with respect to the +1 nt of tRNA. The negative regulatory effect has been narrowed down to a sequence motif 'TATATAA', a perfect consensus recognised by the TATA binding protein, TBP. This sequence element, when brought closer to the transcription start point, on the other hand, exerts a positive effect by promoting transcription of the gene devoid of other cis regulatory elements. The identity of the nuclear protein interacting with this 'TATATAA' element to TBP has been established by antibody and mutagenesis studies. The 'TATATAA' element thus influences the transcription of tRNA genes positively or negatively in a position-dependent manner either by recruitment or sequestration of TBP from the transcription machinery.

Genome-Wide Gene Expression Analysis Reveals a Dynamic Interplay between Luteotropic and Luteolytic Factors in the Regulation of Corpus Luteum Function in the Bonnet Monkey (Macaca radiata)

Although LH is essential for survival and function of the corpus luteum (CL) in higher primates, luteolysis occurs during nonfertile cycles without a discernible decrease in circulating LH levels. Using genome-wide expression analysis, several experiments were performed to examine the processes of luteolysis and rescue of luteal function in monkeys. Induced luteolysis with GnRH receptor antagonist (Cetrorelix) resulted in differential regulation of 3949 genes, whereas replacement with exogenous LH (Cetrorelix plus LH) led to regulation of 4434 genes (1563 down-regulation and 2871 up-regulation). A model system for prostaglandin (PG) F-2 alpha-induced luteolysis in the monkey was standardized and demonstrated that PGF(2 alpha) regulated expression of 2290 genes in the CL. Analysis of the LH-regulated luteal transcriptome revealed that 120 genes were regulated in an antagonistic fashion by PGF(2 alpha). Based on the microarray data, 25 genes were selected for validation by real-time RT-PCR analysis, and expression of these genes was also examined in the CL throughout the luteal phase and from monkeys treated with human chorionic gonadotropin (hCG) to mimic early pregnancy. The results indicated changes in expression of genes favorable to PGF(2 alpha) action during the late to very late luteal phase, and expressions of many of these genes were regulated in an opposite manner by exogenous hCG treatment. Collectively, the findings suggest that curtailment of expression of downstream LH-target genes possibly through PGF(2 alpha) action on the CL is among the mechanisms underlying cross talk between the luteotropic and luteolytic signaling pathways that result in the cessation of luteal function, but hCG is likely to abrogate the PGF(2 alpha)-responsive gene expression changes resulting in luteal rescue crucial for the maintenance of early pregnancy. (Endocrinology 150: 1473-1484, 2009)

Gene patent practice across plant and human genomes

The uses of genetic sequences to inform, enable or create products or services for human biomedicine are substantially different from their uses in crop-based agriculture. Here, we explore what similarities and differences may emerge in patent use and strategies, and map patent-disclosed sequences onto three important plant genomes: maize (corn), rice and soybean. We focus on those referenced in the granted patent claims to compare their uses to the approach used in human gene patenting.

The ownership question of plant gene and genome intellectual properties

The restructuring of the crop agriculture industry over the past two decades has enabled patent holders to exclude, prevent and deter others from using certain research tools and delay or block further follow-on inventions

Structures of three different neutral polysaccharides of Acinetobacter baumannii, NIPH190, NIPH201, and NIPH615, assigned to K30, K45, and K48 capsule types, respectively, based on capsule biosynthesis gene clusters

Neutral capsular polysaccharides (CPSs) were isolated from Acinetobacter baumannii NIPH190, NIPH201, and NIPH615. The CPSs were found to contain common monosaccharides only and to be branched with a side-chain 1→3-linked β-d-glucopyranose residue. Structures of the oligosaccharide repeat units (K units) of the CPSs were elucidated by 1D and 2D 1H and 13C NMR spectroscopy. Novel CPS biosynthesis gene clusters, designated KL30, KL45, and KL48, were found at the K locus in the genome sequences of NIPH190, NIPH201, and NIPH615, respectively. The genetic content of each gene cluster correlated with the structure of the CPS unit established, and therefore, the capsular types of the strains studied were designated as K30, K45, and K48, respectively. The initiating sugar of each K unit was predicted, and glycosyltransferases encoded by each gene cluster were assigned to the formation of the linkages between sugars in the corresponding K unit.

Roles of forced nicotine exposure and Comt gene disruption in the development of addiction-related behavioural and neurochemical changes in mice

Activation of midbrain dopamine systems is thought to be critically involved in the addictive properties of abused substances. Drugs of abuse increase dopamine release in the nucleus accumbens and dorsal striatum, which are the target areas of mesolimbic and nigrostriatal dopamine pathways, respectively. Dopamine release in the nucleus accumbens is thought to mediate the attribution of incentive salience to rewards, and dorsal striatal dopamine release is involved in habit formation. In addition, changes in the function of prefrontal cortex (PFC), the target area of mesocortical dopamine pathway, may skew information processing and memory formation such that the addict pays an abnormal amount of attention to drug-related cues. In this study, we wanted to explore how long-term forced oral nicotine exposure or the lack of catechol-O-methyltransferase (COMT), one of the dopamine metabolizing enzymes, would affect the functioning of these pathways. We also wanted to find out how the forced nicotine exposure or the lack of COMT would affect the consumption of nicotine, alcohol, or cocaine. First, we studied the effect of forced chronic nicotine exposure on the sensitivity of dopamine D2-like autoreceptors in microdialysis and locomotor activity experiments. We found that the sensitivity of these receptors was unchanged after forced oral nicotine exposure, although an increase in the sensitivity was observed in mice treated with intermittent nicotine injections twice daily for 10 days. Thus, the effect of nicotine treatment on dopamine autoreceptor sensitivity depends on the route, frequency, and time course of drug administration. Second, we investigated whether the forced oral nicotine exposure would affect the reinforcing properties of nicotine injections. The chronic nicotine exposure did not significantly affect the development of conditioned place preference to nicotine. In the intravenous self-administration paradigm, however, the nicotine-exposed animals self-administered nicotine at a lower unit dose than the control animals, indicating that their sensitivity to the reinforcing effects of nicotine was enhanced. Next, we wanted to study whether the Comt gene knock-out animals would be a suitable model to study alcohol and cocaine consumption or addiction. Although previous work had shown male Comt knock-out mice to be less sensitive to the locomotor-activating effects of cocaine, the present study found that the lack of COMT did not affect the consumption of cocaine solutions or the development of cocaine-induced place preference. However, the present work did find that male Comt knock-out mice, but not female knock-out mice, consumed ethanol more avidly than their wild-type littermates. This finding suggests that COMT may be one of the factors, albeit not a primary one, contributing to the risk of alcoholism. Last, we explored the effect of COMT deficiency on dorsal striatal, accumbal, and prefrontal cortical dopamine metabolism under no-net-flux conditions and under levodopa load in freely-moving mice. The lack of COMT did not affect the extracellular dopamine concentrations under baseline conditions in any of the brain areas studied. In the prefrontal cortex, the dopamine levels remained high for a prolonged time after levodopa treatment in male, but not female, Comt knock-out mice. COMT deficiency induced accumulation of 3,4-dihydroxyphenylacetic acid, which increased further under levodopa load. Homovanillic acid was not detectable in Comt knock-out animals either under baseline conditions or after levodopa treatment. Taken together, the present results show that although forced chronic oral nicotine exposure affects the reinforcing properties of self-administered nicotine, it is not an addiction model itself. COMT seems to play a minor role in dopamine metabolism and in the development of addiction under baseline conditions, indicating that dopamine function in the brain is well-protected from perturbation. However, the role of COMT becomes more important when the dopaminergic system is challenged, such as by pharmacological manipulation.

Cloning Of Rice Dna And Identification Of Transfer-Rna Gene Clones

THE rapid development of recombinant DNA technology has brought forth a revolution in biology'>", it aids us to have a closer look at the 'way genes are organized, eS11 ecially in the complex eucaryotic genornes'<", Although many animal and yeast genes have been studied in detail using recombinant DNA technology, plant genes have seldom been targets for such studie., Germination is an ideal process to study gene expression .because it effects a . shift in the metabolic status of seeds from a state of 'dormancy to an active one. AJ;l understanding of gene organization and regulation darin.g germination can be accomplblted by molecular cloning of DNA from seeds lik.e rice. To study the status of histone, rRNA tRNA and other genes in the rice genome, a general method was developed to clone eucarvotic DNA in a' plasmid vector pBR 322. This essentially ~ involves the following steps. The rice embryo and plasmid pBR 322 DNAs were cut witll restriction endonuclease Bam Hi to generate stick.Y ends, The plasmid DNA was puosphatased, the DNA~ ware a~·tnealed and joined 'by T4 phage DNA ligase. The recombinant DNA molecules thus produced were transjerred into E. coli and colonies containing them Were selected by their sensitivity to tetracycline and resistance to ampicillin, Two clones were identified . 2S haVing tRNA genes by hybridization of the DNA in the clones \vitl1 32P-la.belled rice tRNAs.