Showing papers in "Molecular Biotechnology in 2011"
TL;DR: The results suggest that miRNAs may play an important role in plant response to environmental abiotic stresses and has the potential to create a miRNA-based biotechnology for improving plant tolerance to drought and salinity stresses.
Abstract: Drought and salinity stresses significantly altered microRNA (miRNA) expression in a dose-dependent manner in tobacco. Salinity stress changed the miRNA expression levels from a 6.86-fold down-regulation to a 616.57-fold up-regulation. Alternatively, miRNAs were down-regulated by 2.68-fold and up-regulated 2810-fold under drought conditions. miR395 was most sensitive to both stresses and was up-regulated by 616 and 2810-folds by 1.00% PEG and 0.171 M NaCl, respectively. Salinity and drought stresses also changed the expression of protein-coding genes [alcohol dehydrogenase (ADH) and alcohol peroxidase (APX)]. The results suggest that miRNAs may play an important role in plant response to environmental abiotic stresses. Further investigation of miRNA-mediated gene regulation may elucidate the molecular mechanism of plant tolerance to abiotic stresses and has the potential to create a miRNA-based biotechnology for improving plant tolerance to drought and salinity stresses.
173 citations
TL;DR: The catalytic subunit of protein phosphatase 2A was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation; the commonly used reference gene actin was generally the least stable of the set.
Abstract: Quantitative real-time PCR (RT-qPCR) is a reliable method for assessing gene expression, provided that suitable reference genes are included to normalize the data. The stability of expression of eight potential reference genes, namely, tubulin (alpha-2,4 tubulin), actin, EF1 α (elongation factor 1 α), UBC (ubiquitin C), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), psaA (photosynthesis-related plastid gene representing photosystem I), PP2Acs (catalytic subunit of protein phosphatase 2A), and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to aphid infestation, heat stress or waterlogging stress using geNorm software. The widely used reference gene EF1 α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase 2A (PP2Acs) was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. No single gene was suitable for normalization on its own. The choice of reference gene(s) is an important factor in gene expression studies based on RT-qPCR.
124 citations
TL;DR: A rapid and efficient nucleic acids extraction protocol that in contrast to other methods tested, effectively purify high qualityucleic acids from plant tissues rich in polysaccharides and polyphenolic compounds such as different grape tissues and fruit tissue of fruit trees is described.
Abstract: Isolation of high quality nucleic acids from plant tissues rich in polysaccharides and polyphenols is often difficult. The presence of these substances can affect the quality and/or quantity of the nucleic acids isolated. Here, we describe a rapid and efficient nucleic acids extraction protocol that in contrast to other methods tested, effectively purify high quality nucleic acids from plant tissues rich in polysaccharides and polyphenolic compounds such as different grape tissues and fruit tissue of fruit trees. The nucleic acids isolated with this protocol were successfully used for many functional genomic based experiments including polymerase chain reaction, reverse transcription polymerase chain reaction (RT-PCR), cloning, and semiquantitative RT-PCR.
115 citations
TL;DR: High throughput screening (HTS) is at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics.
Abstract: High throughput screening (HTS) is at the core of the drug discovery process, and so it is critical to design and implement HTS assays in a comprehensive fashion involving scientists from the disciplines of biology, chemistry, engineering, and informatics. This requires careful analysis of many variables, starting with the choice of assay target and ending with the discovery of lead compounds. At every step in this process, there are decisions to be made that can greatly impact the outcome of the HTS effort, to the point of making it a success or a failure. Although specific guidelines should be established to insure that the screening assay reaches an acceptable level of quality, many choices require pragmatism and the ability to compromise opposing forces.
98 citations
TL;DR: It was found that transgenic plants having AtNHX1 gene are more resistant to high concentration of salt and water deprivation than the wild type plants and could be cultivated in salt and drought-affected soils.
Abstract: Salinity and drought are main threat to agriculture productivity, to avoid further losses it is necessary to improve the genetic material of crops against these stresses In this present study, AtNHX1, a vacuolar type Na+/H+ antiporter gene driven by 35S promoter was introduced into groundnut using Agrobacterium tumefaciens transformation system. The stable integration of the AtNHX1 gene was confirmed by polymerase chain reaction (PCR) and southern blot analysis. It was found that transgenic plants having AtNHX1 gene are more resistant to high concentration of salt and water deprivation than the wild type plants. Salt and proline level in the leaves of the transgenic plants were also much higher than that of wild type plants. The results showed that overexpression of AtNHX1 gene not only improved salt tolerance but also drought tolerance in transgenic groundnut. Our results suggest that these plants could be cultivated in salt and drought-affected soils.
92 citations
TL;DR: Results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants.
Abstract: The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A transcript encoding NAC protein, termed SiNAC was identified from a salt stress subtractive cDNA library of S. italica seedling (Puranik et al., J Plant Physiol 168:280–287, 2011). This single/low copy gene containing four exons and four introns within the genomic-sequence encoded a protein of 462 amino acids. Structural analysis revealed that highly divergent C terminus contains a transmembrane domain. The NAC domain consisted of a twisted antiparallel beta-sheet packing against N terminal alpha helix on one side and a shorter helix on the other side. The domain was predicted to homodimerize and control DNA-binding specificity. The physicochemical features of the SiNAC homodimer interface justified the dimeric form of the predicted model. A 1539 bp fragment upstream to the start codon of SiNAC gene was cloned and insilico analysis revealed several putative cis-acting regulatory elements within the promoter sequence. Transactivation analysis indicated that SiNAC activated expression of reporter gene and the activation domain lied at the C terminal. The SiNAC:GFP was detected in the nucleus and cytoplasm while SiNAC ΔC1−158:GFP was nuclear localized in onion epidermal cells. SiNAC transcripts mostly accumulated in young spikes and were strongly induced by dehydration, salinity, ethephon, and methyl jasmonate. These results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants.
85 citations
TL;DR: Some of the key observations that support biological functions for G-quadruplex DNA as well as the techniques and tools that have enabled researchers to probe these structures and their interactions with proteins and small molecules are reviewed.
Abstract: G-quartets are square planar arrangements of four guanine bases, which can form extraordinarily stable stacks when present in nucleic acid sequences. Such G-quadruplex structures were long regarded as an in vitro phenomenon, but the widespread presence of suitable sequences in genomes and the identification of proteins that stabilize, modify or resolve these nucleic acid structures have provided circumstantial evidence for their physiological relevance. The therapeutic potential of small molecules that can stabilize or disrupt G-quadruplex structures has invigorated the field in recent years. Here we review some of the key observations that support biological functions for G-quadruplex DNA as well as the techniques and tools that have enabled researchers to probe these structures and their interactions with proteins and small molecules.
75 citations
TL;DR: As proteomic information reflects a direct view of cellular systems, proteomics is expected to complement other areas of biology such as genomics, transcriptomics, molecular biology, cloning, and classical genetics in understanding the relationships among multiple facets of biological systems.
Abstract: In the recent years, research in molecular biotechnology has transformed from being small scale studies targeted at a single or a small set of molecule(s) into a combination of high throughput discovery platforms and extensive validations. Such a discovery platform provided an unbiased approach which resulted in the identification of several novel genetic and protein biomarkers. High throughput nature of these investigations coupled with higher sensitivity and specificity of Next Generation technologies provided qualitatively and quantitatively richer biological data. These developments have also revolutionized biological research and speed of data generation. However, it is becoming difficult for individual investigators to directly benefit from this data because they are not easily accessible. Data resources became necessary to assimilate, store and disseminate information that could allow future discoveries. We have developed two resources—Human Protein Reference Database (HPRD) and Human Proteinpedia, which integrate knowledge relevant to human proteins. A number of protein features including protein–protein interactions, post-translational modifications, subcellular localization, and tissue expression, which have been studied using different strategies were incorporated in these databases. Human Proteinpedia also provides a portal for community participation to annotate and share proteomic data and uses HPRD as the scaffold for data processing. Proteomic investigators can even share unpublished data in Human Proteinpedia, which provides a meaningful platform for data sharing. As proteomic information reflects a direct view of cellular systems, proteomics is expected to complement other areas of biology such as genomics, transcriptomics, molecular biology, cloning, and classical genetics in understanding the relationships among multiple facets of biological systems.
73 citations
TL;DR: Results suggest that Agrobacteriumtumefaciens-mediated transformation can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.
Abstract: Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacteriumtumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.
72 citations
TL;DR: The study showed that the region, RM212–RM302–RM8085–RM3825 on chromosome 1, harbours large effect QTLs for drought-resistance traits across several genetic backgrounds in rice, which may be useful for drought resistance improvement in rice through MAS and map-based cloning.
Abstract: Drought stress is a major limitation to rice (Oryza sativa L.) yields and its stability, especially in rainfed conditions. Developing rice cultivars with inherent capacity to withstand drought stress would improve rainfed rice production. Mapping quantitative trait loci (QTLs) linked to drought resistance traits will help to develop rice cultivars suitable for water-limited environments through molecular marker-assisted selection (MAS) strategy. However, QTL mapping is usually carried out by genotyping large number of progenies, which is labour-intensive, time-consuming and cost-ineffective. Bulk segregant analysis (BSA) serves as an affordable strategy for mapping large effect QTLs by genotyping only the extreme phenotypes instead of the entire mapping population. We have previously mapped a QTL linked to leaf rolling and leaf drying in recombinant inbred (RI) lines derived from two locally adapted indica rice ecotypes viz., IR20/Nootripathu using BSA. Fine mapping the QTL will facilitate its application in MAS. BSA was done by bulking DNA of 10 drought-resistant and 12 drought-sensitive RI lines. Out of 343 rice microsatellites markers genotyped, RM8085 co-segregated among the RI lines constituting the respective bulks. RM8085 was mapped in the middle of the QTL region on chromosome 1 previously identified in these RI lines thus reducing the QTL interval from 7.9 to 3.8 cM. Further, the study showed that the region, RM212–RM302–RM8085–RM3825 on chromosome 1, harbours large effect QTLs for drought-resistance traits across several genetic backgrounds in rice. Thus, the QTL may be useful for drought resistance improvement in rice through MAS and map-based cloning.
66 citations
TL;DR: The results indicate that ABA-GA antagonism is a key focal point for understanding the failure of panicle exsertion under drought stress and the consequent increase in spikelet sterility.
Abstract: Panicle exsertion, an essential physiological process for obtaining high grain yield in rice is mainly driven by peduncle (uppermost internode) elongation. Drought at heading/panicle emergence prevented peduncle elongation from reaching its maximum length even after re-watering. This inhibitory effect of drought resulted in delayed heading and trapping spikelets lower down the panicle inside the flag-leaf sheath, thus increasing sterility in the lower un-exserted spikelets and also among the upper superior spikelets whose exsertion was delayed. Intermittent drought stress caused a significant reduction in relative water content (RWC) and an increase in the abscisic acid (ABA) level of the peduncles, while both returned to normal levels upon re-watering. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis revealed the down-regulation of GA biosynthetic genes during drought. 2D-PAGE analysis of proteins from peduncles collected under well-watered, drought-stressed, and re-watered plants revealed at least twofold differential changes in expression of 31 proteins in response to drought and most of these changes were largely reversed by re-watering. The results indicate that ABA-GA antagonism is a key focal point for understanding the failure of panicle exsertion under drought stress and the consequent increase in spikelet sterility.
TL;DR: It is demonstrated that two MYMIV-resistance marker loci could be efficiently employed together in a multiplex-PCR-reaction for genotyping both V. mungo and V. radiata germplasms from field grown plants and also directly from the seed stock.
Abstract: Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus begomovirus causes the yellow mosaic disease in a number of economically important edible grain legumes including mungbean (Vigna radiata), urdbean (Vigna mungo) and soybean (Glycine max). The disease is severe, critical, open spread and inflicts heavy yield losses annually. The objective of this study is to develop molecular markers linked to MYMIV-resistance to facilitate genotyping of urdbean and mungbean germplasms for MYMIV-reaction. Resistance-linked molecular markers were successfully developed from consensus motifs of other resistance (R) gene or R gene homologue sequences. Applying linked marker-assisted genotyping, plant breeders can carry out repeated genotyping throughout the growing season in absence of any disease incidence. Two MYMIV-resistance marker loci, YR4 and CYR1, were identified and of these two CYR1 is completely linked with MYMIV-resistant germplasms and co-segregating with MYMIV-resistant F2, F3 progenies of urdbean. The present study demonstrated that these two markers could be efficiently employed together in a multiplex-PCR-reaction for genotyping both V. mungo and V. radiata germplasms from field grown plants and also directly from the seed stock. This method of genotyping would save time and labour during the introgression of MYMIV-resistance through molecular breeding, as methods of phenotyping against begomoviruses are tedious, labour and time intensive.
TL;DR: The sequence-related amplified polymorphism (SRAP) technique, aimed for the amplification of open reading frames (ORFs), vis-â-vis that of the amplified fragment length polymorphisms (AFLP) were used to analyze the genetic variation and relationships among forty Musa accessions.
Abstract: The sequence-related amplified polymorphism (SRAP) technique, aimed for the amplification of open reading frames (ORFs), vis-â-vis that of the amplified fragment length polymorphisms (AFLP) were used to analyze the genetic variation and relationships among forty Musa accessions; which include commercial cultivars and wild species of interest for the genetic enhancement of Musa. A total of 403 SRAP and 837 AFLP amplicons were generated by 10 SRAP and 15 AFLP primer combinations, of which 353 and 787 bands were polymorphic, respectively. Both cluster analysis of unweighted pair-grouping method with arithmetic averages (UPGMA) and principal coordinate (PCO) analysis separated the forty accessions into their recognized sections (Eumusa, Australimusa, Callimusa and Rhodochlamys) and species. The percentage of polymorphism amongst sections and species and the relationships within Eumusa species and subspecies varied between the two marker systems. In addition to its practical simplicity, SRAP exhibited approximately threefold more specific and unique bands than AFLP, 37 and 13%, respectively. SRAP markers are demonstrated here to be proficient tools for discriminating amongst M. acuminata, M. balbisiana and M. schizocarpa in the Eumusa section, as well as between plantains and cooking bananas within triploid cultivars.
TL;DR: This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it, and allows rapid pathogen detection.
Abstract: Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.
TL;DR: Correspondence analysis of relative synonymous codon usages showed that genes on secondary axis were correlated with their GC3S values, indicating mutational bias as an important selective force that shaped the variation in the codon usage among chloroplast genes.
Abstract: We have analyzed factors affecting the codon usage pattern of the chloroplasts genomes of representative species of pooid grass family. Correspondence analysis of relative synonymous codon usages (RSCU) showed that genes on secondary axis were correlated with their GC3S values (all r > 0.3, p < 0.05), indicating mutational bias as an important selective force that shaped the variation in the codon usage among chloroplast genes. The Nc-plot showed that although a majority of the points with low-Nc values were lying below the expected curve, a few genes lied on the expected curve. Nc plot clearly showed that mutational bias plays a major role in codon biology across the monocot plastomes. The hydrophobicity and aromaticity of encoded proteins of each species were found to be other factors of codon usage variation. In the view of above light, besides natural selection, several other factors also likely to be involved in determining the selective constraints on codon bias in plastomes of pooid grass genomes. In addition, five codons (B. distachyon), seven codons (H. vulgare), and four codons (T. aestivum) were identified as optimal codons of the three grass chloroplasts. To identify genes evolving under positive selection, rates of nonsynonymous substitutions (Ka) and synonymous substitutions (Ks) were computed for all groups of orthologous gene pairs.
TL;DR: The results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast and in vivo polysome levels and protein synthesis are analyzed as a whole.
Abstract: In an effort to develop microalgae as a robust system for the production of valuable proteins, we analyzed some of the factors affecting recombinant protein expression in the chloroplast of the green alga Chlamydomonas reinhardtii. We monitored mRNA accumulation, protein synthesis, and protein turnover for three codon-optimized transgenes including GFP, bacterial luciferase, and a large single chain antibody. GFP and luciferase proteins were quite stable, while the antibody was less so. Measurements of protein synthesis, in contrast, clearly showed that translation of the three chimeric mRNAs was greatly reduced when compared to endogenous mRNAs under control of the same atpA promoter/UTR. Only in a few conditions this could be explained by limited mRNA availability since, in most cases, recombinant mRNAs accumulated quite well when compared to the atpA mRNA. In vitro toeprint and in vivo polysome analyses suggest that reduced ribosome association might contribute to limited translational efficiency. However, when recombinant polysome levels and protein synthesis are analyzed as a whole, it becomes clear that other steps, such as inefficient protein elongation, are likely to have a considerable impact. Taken together, our results point to translation as the main step limiting the expression of heterologous proteins in the C. reinhardtii chloroplast.
TL;DR: Dendrograms based on three molecular data using unweighted pair group method with arithmetic mean was congruent and classified the Curcuma species into two major clusters and was further supported by principle coordinate analysis.
Abstract: Molecular genetic fingerprints of nine Curcuma species from Northeast India were developed using PCR-based markers. The aim involves elucidating there intra- and inter-specific genetic diversity important for utilization, management, and conservation. Twelve random amplified polymorphic DNA (RAPD), 19 Inter simple sequence repeats (ISSRs), and four amplified fragment length polymorphism (AFLP) primers produced 266 polymorphic fragments. ISSR confirmed maximum polymorphism of 98.55% whereas RAPD and AFLP showed 93.22 and 97.27%, respectively. Marker index and polymorphic information content varied in the range of 8.64–48.1, 19.75–48.14, and 25–28 and 0.17–0.48, 0.19–0.48, and 0.25–0.29 for RAPD, ISSR, and AFLP markers, respectively. The average value of number of observed alleles, number of effective alleles, mean Nei’s gene diversity, and Shannon’s information index were 1.93–1.98, 1.37–1.62, 0.23–0.36, and 0.38–0.50, respectively, for three DNA markers used. Dendrograms based on three molecular data using unweighted pair group method with arithmetic mean (UPGMA) was congruent and classified the Curcuma species into two major clusters. Cophenetic correlation coefficient between dendrogram and original similarity matrix were significant for RAPD (r = 0.96), ISSR (r = 0.94), and AFLP (r = 0.97). Clustering was further supported by principle coordinate analysis. High genetic polymorphism documented is significant for conservation and further improvement of Curcuma species.
TL;DR: Transgenic lettuce successfully expressing CTB–COE fusion protein will be tested to induce efficient immune responses against porcine epidemic diarrhea virus infection by administration with raw material.
Abstract: Transgenic plants have been used as a safe and economic expression system for the production of edible vaccines. A synthetic cholera toxin B subunit gene (CTB) was fused with a synthetic neutralizing epitope gene of the porcine epidemic diarrhea virus (sCTB–sCOE), and the sCTB–sCOE fusion gene was introduced into a plant expression vector under the control of the ubiquitin promoter. This plant expression vector was transformed into lettuce (Lactuca sativa L.) using the Agrobacterium-mediated transformation method. Stable integration and transcriptional expression of the sCTB–sCOE fusion gene was confirmed using genomic DNA PCR analysis and northern blot analysis, respectively. The results of western blot analysis with anti-cholera toxin and anti-COE antibody showed the synthesis and assembly of CTB–COE fusion protein into oligomeric structures with pentameric sizing. The biological activity of CTB–COE fusion protein to its receptor, GM1-ganglioside, in transgenic plants was confirmed via GM1-ELISA with anti-cholera toxin and anti-COE antibody. Based on GM1-ELISA, the expression level of CTB–COE fusion proteins reached 0.0065% of the total soluble protein in transgenic lettuce leaf tissues. Transgenic lettuce successfully expressing CTB–COE fusion protein will be tested to induce efficient immune responses against porcine epidemic diarrhea virus infection by administration with raw material.
TL;DR: The OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants and Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development.
Abstract: Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.
TL;DR: The results demonstrate the Epi-CHO system’s capacity for the rapid production of CHO cell-derived recombinant monoclonal antibodies in serum-free conditions and higher transgene mRNA and protein levels compared to cell lines deficient in plasmid DNA replication and/or retention post-transfection.
Abstract: Transient gene expression systems in mammalian cells continue to grow in popularity due to their capacity to produce significant amounts of recombinant protein in a rapid and scalable manner, without the lengthy time periods and resources required for stable cell line development. Traditionally, production of recombinant monoclonal antibodies for pre-clinical assessment by transient expression in CHO cells has been hampered by low titers. In this report, we demonstrate transient monoclonal antibody titers of 140 mg/l with CHO cells using the episomal-based transient expression system, Epi-CHO. Such titers were achieved by implementing an optimized transfection protocol incorporating mild-hypothermia and through screening of a variety of chemically defined and serum-free media for their ability to support elevated and prolonged viable cell densities post-transfection, and in turn, improve recombinant protein yields. Further evidence supporting Epi-CHO’s capacity to enhance transgene expression is provided, where we demonstrate higher transgene mRNA and protein levels of two monoclonal antibodies and a destabilized enhanced green fluorescent protein with Epi-CHO compared to cell lines deficient in plasmid DNA replication and/or retention post-transfection. The results demonstrate the Epi-CHO system’s capacity for the rapid production of CHO cell-derived recombinant monoclonal antibodies in serum-free conditions.
TL;DR: Focus of this review is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimisation for kinases and high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological samples.
Abstract: Enzymes are key molecules in signal-transduction pathways However, only a small fraction of more than 500 human kinases, 300 human proteases and 200 human phosphatases is characterised so far Peptide microarray based technologies for extremely efficient profiling of enzyme substrate specificity emerged in the last years This technology reduces set-up time for HTS assays and allows the identification of downstream targets Moreover, peptide microarrays enable optimisation of enzyme substrates Focus of this review is on assay principles for measuring activities of kinases, phosphatases or proteases and on substrate identification/optimisation for kinases Additionally, several examples for reliable identification of substrates for lysine methyl-transferases, histone deacetylases and SUMO-transferases are given Finally, use of high-density peptide microarrays for the simultaneous profiling of kinase activities in complex biological samples like cell lysates or lysates of complete organisms is described All published examples of peptide arrays used for enzyme profiling are summarised comprehensively
TL;DR: Thermo-stability of FLP recombinase in rice cells is critical for efficient site-specific recombination, and use of FLPe offers practical solutions to FLP–FRT-based biotechnology applications in plants.
Abstract: Site-specific recombination systems, such as FLP–FRT and Cre–lox, carry out precise recombination reactions on their respective targets in plant cells. This has led to the development of two important applications in plant biotechnology: marker-gene deletion and site-specific gene integration. To draw benefits of both applications, it is necessary to implement them in a single transformation process. In order to develop this new process, the present study evaluated the efficiency of FLP–FRT system for excising marker gene from the transgene locus developed by Cre–lox mediated site-specific integration in rice. Two different FLP recombinases, the wild-type FLP (FLPwt) and its thermostable derivative, FLPe, were used for the excision of marker gene flanked by FLP recombination targets (FRT). While marker excision mediated by FLPwt was undetectable, use of FLPe resulted in efficient marker excision in a number of transgenic lines, with the relative efficiency reaching up to ~100%. Thus, thermo-stability of FLP recombinase in rice cells is critical for efficient site-specific recombination, and use of FLPe offers practical solutions to FLP–FRT-based biotechnology applications in plants.
TL;DR: Results clearly demonstrated that Tween 80 treatment permeabilized the roots to enhance secretion, but also acted as an efficient elicitor of licochalcone A and total flavonoid production in hairy roots of G. uralensis Fisch.
Abstract: We evaluated the effect of Tween 80 as elicitor on licochalcone A from hairy root cultures of Glycyrrhiza uralensis Fisch. After a 15-days treatment with 2% Tween 80, hairy roots still grew well and produced higher levels of licochalcone A and total flavonoids than the control (without treatment). Licochalcone A content and total flavonoid content were 3.103 and 127.095 mg per flask (9- and 11-fold higher), respectively, compared with controls. Secretion of licochalcone A and total flavonoids into the culture medium was remarkably high, up to 98 and 94% of the total production, respectively. The enhanced flavonoid production was associated with elevated mRNA levels and enzyme activities of phenylalanine ammonia-lyase (PAL), 4-coumarate:coenzyme A ligase (4CL), and cinnamate-4-hydroxylase (C4H). These results clearly demonstrated that Tween 80 treatment permeabilized the roots to enhance secretion, but also acted as an efficient elicitor of licochalcone A and total flavonoid production in hairy roots of G. uralensis Fisch.
TL;DR: Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures, and it was found to work equally well for isolating RNA from other desert plants, likely to be widely applicable.
Abstract: RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.
TL;DR: Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition.
Abstract: Lentiviral-mediated gene transfer in vivo or in cultured mammalian neurons can be used to address a wide variety of biological questions, to design animals models for specific neurodegenerative pathologies, or to test potential therapeutic approaches in a variety of brain disorders. Lentiviruses can infect non-dividing cells, thereby allowing stable gene transfer in post-mitotic cells such as mature neurons. An important contribution has been the use of inducible vectors: the same animal can thus be used repeatedly in the doxycycline-on or -off state, providing a powerful mean for assessing the function of a gene candidate in a disorder within a specific neuronal circuit. Furthermore, lentivirus vectors provide a unique tool to integrate siRNA expression constructs with the aim to locally knockdown expression of a specific gene, enabling to assess the function of a gene in a very specific neuronal pathway. Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in the brain. Therefore, the use of lentiviruses for stable expression of siRNA in brain is a powerful aid to probe gene functions in vivo and for gene therapy of diseases of the central nervous system. In this chapter I review the applications of lentivirus-mediated gene transfer in the investigation of specific gene candidates involved in major brain disorders and neurodegenerative processes. Major applications have been in polyglutamine disorders, such as synucleinopathies and Parkinson’s disease, or in investigating gene function in Huntington’s disease, dystonia, or muscular dystrophy. Recently, lentivirus gene transfer has been an invaluable tool for evaluation of gene function in behavioral disorders such as drug addiction and attention-deficit hyperactivity disorder or in learning and cognition.
TL;DR: The results indicate that demography may influence the diversity of the repertoire of the library in terms of its capacity to generate antibodies to specific targets, and this is linked to its intrinsic diversity, which exceeds many existing conventional human libraries.
Abstract: A unique human phage display library was used to successfully generate a scFv to the highly carcinogenic toxin aflatoxin B1 Such an antibody has major potential applications in therapy and diagnostics To further exploit its analytical capacity, the scFv was genetically fused to alkaline phosphatase, thereby generating a novel and highly sensitive self-indicating reagent The performance of this reagent was further characterized, demonstrating its efficacy The sensitivity of scFv-AP fusion was three-fold better than that of the scFv form The ability of this human library to generate antibodies to a small hapten was clearly demonstrated and this is linked to its intrinsic diversity, which exceeds many existing conventional human libraries Our results indicate that demography may influence the diversity of the repertoire of the library in terms of its capacity to generate antibodies to specific targets Equally, the approach demonstrated should also be applicable for other haptens and larger antigens
TL;DR: Genipin cross-linked polymeric microcapsules can emerge as a potentially important tool for improved stem cell-based therapy and cell delivery applications because of its biocompatible and bioactive attributes.
Abstract: The ability of mesenchymal stem cells to self-renew and differentiate into specialized cell lineages makes them promising tools for regenerative medicine. Local injection and use of scaffolds had been employed earlier to deliver these cells; yet, an optimal delivery system remains to be identified. Here, using genipin, which is a non-toxic natural cross linker for proteins, we prepared alginate–chitosan polymeric microcapsules (GCAC) to develop an efficient stem cell delivery system. We investigated the properties of this membrane along with the encapsulated adipose tissue-derived stem cells (ASCs) and compared that with the widely used alginate poly-lysine (APA) membranes. The GCAC membrane was able to support cell viability, augment cell growth, and showed better results under external rotational and osmotic pressures with about 30% of the ruptured capsules in comparison to 60% ruptured APA capsules. The membrane also provided immune-protection to the entrapped cells as demonstrated by the lymphocyte proliferation assay. The capsule also has potential for long-term storage. The encapsulated four million ASCs also showed steady secretion of approximately 4600 pg vascular endothelial growth factor (VEGF) over 15-day time period comparable to that of free cells. Furthermore, the encapsulated ASCs showed around 3.8-fold increase in VEGF secretion after 72 h hypoxic conditions in comparison to normoxic conditions. This increased VEGF expression resulted in improved angiogenic potential of the bioactive capsules as noted by enhanced endothelial cell growth. GCAC encapsulation also did not show any effect on their differentiation ability. Thus, because of these biocompatible and bioactive attributes, genipin cross-linked polymeric microcapsules can emerge as a potentially important tool for improved stem cell-based therapy and cell delivery applications.
TL;DR: Murine IgG1, the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 1011 affibody molecules, resulting in the identification of binders with broad mIgG1 recognition and dissociation constants in the low nanomolar to low micromolar range.
Abstract: Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG1, the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 1011 affibody molecules. Four rounds of selection using three different mouse IgG1 mAbs as alternating targets resulted in the identification of binders with broad mIgG1 recognition and dissociation constants (KD) in the low nanomolar to low micromolar range. For one of the binders, denoted Zmab25, competition in binding to full length mIgG1 by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG1 Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG1 had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG1 nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Zmab25. This selective binding profile towards murine IgG1 was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.
TL;DR: The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system.
Abstract: Macroporous microcarriers entrap cells in a mesh network allowing growth to high densities and protect them from high shear forces in stirred bioreactor cultures. We report the growth of Chinese hamster ovary (CHO) cells producing either recombinant human beta-interferon (β-IFN) or recombinant human tissue-plasminogen activator (t-PA) in suspension or embedded in macroporous microcarriers (Cytopore 1 or 2). The microcarriers enhanced the volumetric production of both β-IFN and t-PA by up to 2.5 fold compared to equivalent suspension cultures of CHO cells. Under each condition the cell specific productivity (Q
P) was determined as units of product/cell per day based upon immunological assays. Cells grown in Cytopore 1 microcarriers showed an increase in Q
P with increasing cell densities up to a threshold of >1 × 108 cells/ml. At this point the specific productivity was 2.5 fold higher than equivalent cells grown in suspension but cell densities above this threshold did not enhance Q
P any further. A positive linear correlation (r
2 = 0.93) was determined between the specific productivity of each recombinant protein and the corresponding cell density for CHO cells grown in Cytopore 2 cultures. With a cell density range of 25 × 106 to 3 × 108 cells/ml within the microcarriers there was a proportional increase in the specific productivity. The highest specific productivity measured from the microcarrier cultures was ×5 that of suspension cultures. The relationship between specific productivity and cell density within the microcarriers leads to higher yields of recombinant proteins in this culture system. This could be attributed to the environment within the microcarrier matrix that may influence the state of cells that could affect protein synthesis or secretion.
TL;DR: A diverse set of vectors are modified to include a broadly expressed hygromycin resistance (HmR) marker and each vector has an origin of transfer for conjugation, and is able to replicate in Saccharomyces cerevisiae to take advantage of the powerful yeast recombineering system.
Abstract: The ability of many bacterial strains to tolerate antibiotics can limit the number of molecular tools available for research of these organisms. To help address this problem, we have modified a diverse set of vectors to include a broadly expressed hygromycin resistance (HmR) marker. Hygromycin B is an aminoglycoside antibiotic not used to treat infections in humans and has antimicrobial activity against a wide range of microorganisms. Vectors with four replication origins are represented, with potential applications including general cloning, allelic replacement, and transcriptional analysis. We show that vectors with the broad host range pBBR1-replicon conferred HmR to Achromobacter xylosoxidans, Acinetobacter baumannii, Pseudomonas aeruginosa, and Serratia marcescens, and a pC194-based vector was able to confer HmR to Francisella tularensis. We also used a subset of these plasmids to manipulate the genome of S. marcescens. Each vector has an origin of transfer for conjugation, and is also able to replicate in Saccharomyces cerevisiae to take advantage of the powerful yeast recombineering system.