A Novel Genotyping Strategy Based on Allele-specific Inverse PCR for Rapid and Reliable Identification of Conditional FADD Knockout Mice

The apoptotic adapter protein FADD has been shown to play diverse roles in cell survival and proliferation. FADD knockout embryos died of heart defects, rendering Cre/loxP-mediated conditional FADD knockout mice a unique tool for investigating FADD-dependent nonapoptotic mechanism. Previously, these genetically engineered mice were identified by time-consuming Southern blot or controversial real-time PCR. In this article, we report a novel genotyping strategy based on allele-specific inverse PCR (ASI-PCR) for rapid and reliable identification of conditional FADD knockout mice. In this strategy, the knockout nature of FADD was simply identified by screening the absence of the wild type FADD-specific ASI-PCR product. Using this method, we accurately identified CD4-Cre-mediated T cell specific FADD knockout mice. The whole process can be accomplished in any normal biological laboratory within 12 h using genomic DNA from tail biopsy. The proposed ASI-PCR-based approach is simple, rapid, sensitive, reproducible, and especially suitable for genotyping small amount of spatiotemporally restricted biopsies and large animal population. We believe that the strategy described in this article may be of general utility in genotyping other conditional gene knockout mice.     

Microbial Biotechnology is 15! An adventure in journal parenting

This year, 2022, Microbial Biotechnology (MBT) celebrates its 15th birthday. In journal terms, 15 is adulthood. It has been a privilege to develop the idea, launch the journal, nurture it in its infancy and stimulate and support it during its adolescence and early adulthood. The success of MBT – its growth over the last 4 years averaging > 30%, and the highest impact factor in the field, making it the leading research journal in applied microbiology/microbial biotechnology and the most attractive to publish in – gives us enormous pleasure and satisfaction, and not a little pride.     

Rapid one-step inactivation of single or multiple genes in Escherichia coli

Gene knockout experiments are frequently performed for both fundamental and applied biological research. We developed an integration helper plasmid-based knockout system for more efficient and rapid engineering of Escherichia coli. The integration helper plasmid, pCW611, contains two recombinases that are expressed in the reverse direction by two independent inducible systems. One is Red recombinase under the control of the arabinose-inducible system to induce a recombination event by using the linear gene knockout DNA fragment, while the other is Cre recombinase, which is controlled by the isopropyl β-D -1-thiogalactopyranoside-inducible system to obtain markerless mutant strains. The time and effort required can be reduced with this system because iterative transformation and curing steps are not required. We could delete one target gene in three days by using pCW611. To verify the usefulness of this system, deletion experiments were performed to knock out four target genes individually (adhE, sfcA, frdABCD, and ackA) and two genes simultaneously for two cases (adhE–aspA and sfcA–aspA). Also, sequential deletion of four target genes (fumB, iclR, fumA, and fumC) was successfully performed to make a fumaric acid producing strain. This successfully developed and validated rapid and efficient gene manipulation system should be useful for the metabolic engineering of E. coli.     

细胞可透过性Cre重组酶表达、纯化及生物活性检测(英)

Cre/lox P系统由Cre位点特异重组酶和可被Cre特异性识别的lox P位点构成,该系统广泛用于条件性基因敲除和表达,以研究基因功能.为了表达和纯化一种细胞可透过性Cre重组酶(即His6-NLS-Cre-MTS);经IPTG诱导,在BL21(DE3)宿主菌成功表达His6-NLS-Cre-MTS融合蛋白,通过His-Bond Ni-NTA树脂分离并纯化了该蛋白质,随后借助细胞和非细胞的重组系统成功检测了His6-NLS-Cre-MTS的生物活性.细胞可透过性Cre重组酶提供了一种快捷而高效的在细胞和在体水平进行遗传操作的新工具.     

Asian Federation of Biotechnology (AFOB)

Cover illustration: This issue is the first official special issue of Biotechnology Journal with the Asian Federation of Biotechnology (AFOB). In the cover, we use the grain as a symbol and analogy for biotechnology, as both the grain and biotechnology are common and uniting factors that link the member countries of the AFOB. Gran image credit: © Myimagine– Fotolia.com.     

Rapid method for simultaneous determination of 20 components in Isodon nervosa by high‐performance liquid chromatography–electrospray ionisation tandem mass spectrometry

Introduction – Isodon nervosa is a commonly used traditional Chinese medicine including diterpenoids, phenolic acids, triterpenoids and volatile oil. Qualitative and quantitative analysis of multi‐components is important for its quality control. Objective – To establish a liquid chromatography–electrospray ionisation–mass spectrometry method for simultaneous analysis of 20 bioactive constituents of Isodon nervosa in different places of China and different parts of this herb. Methodology – The optimal chromatographic conditions were achieved on a C₁₈ column (250 × 4.6 mm, 5 µm) with with linear gradient elution with 0.1% aqueous formic acid : methanol containing 0.1% formic acid at a flow‐rate of 0.7 mL/min in 15 min. The identification and quantification of those analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple‐reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the method was carried out (linearity, precision, accuracy, limit of detection and limit of quantification). Results – The results indicated that the method was simple, rapid, specific and reliable. The proposed method was successfully applied for the qualitative and quantitative analysis of 20 chemical compositions in Isodon nervosa samples. Conclusion – Twenty chemical compositions in 21 batches of wild and cultivated Isodon nervosa samples from different sources had great variation in the contents. Copyright © 2010 John Wiley & Sons, Ltd.     

Litter and aerosol sampling of chicken houses for rapid detection of Salmonella typhimurium contamination using gene amplification

Rapid screening of poultry houses for contamination is critical for Salmonella control. Use of air filter sampling has great potential for efficient and reliable monitoring of Salmonella spp., as it could represent an entire poultry house and solve sample-size problems. Two sampling methods (litter and air filter) were compared for detection in four chicken pens inoculated with a S. typhimurium antibiotic resistant strain. Salmonella levels in both litter and air filter samples were determined by PCR amplification and by conventional enrichment. Although amplified DNA was not directly detected, amplified DNA could be detected using a dual probe hybridization sensor. The ratio of the positive samples to total samples determined by gene amplification was much lower than that obtained by conventional enrichments (29/128 versus 102/128 samples). However, the ratio obtained by gene amplification with air filter samples was greater than that with litter samples (26/64 versus 3/64). These results demonstrate that the air filter sampling method is an alternative method of Salmonella detection in poultry house using PCR gene amplification protocol. Journal of Industrial Microbiology & Biotechnology (2000) 24, 379–382. Received 12 August 1999/ Accepted in revised form 17 February 2000     

Rapid validated HPTLC method for estimation of betulinic acid in Nelumbo nucifera (Nymphaeaceae) rhizome extract

Introduction – Betulinic acid (pentacyclic triterpenoid) is an important marker component present in Nelumbo nucifera Gaertn. rhizome. N. nucifera rhizome has several medicinal uses including hypoglycaemic, antidiarrhoeal, antimicrobial, diuretic, antipyretic, psychopharmacological activities. Objective – To establish a simple, sensitive, reliable, rapid and validated high‐performance thin‐layer chromatography method for estimation of betulinic acid in hydro‐alcoholic extract of N. nucifera Gaertn. rhizome. Materials and methods – The separation was carried out on a thin‐layer chromatography aluminium plate pre‐coated with silica gel 60F₂₅₄, eluted with chloroform, methanol and formic acid (49 : 1 : 1 v/v). Post chromatographic derivatisation was done with anisaldehyde–sulphuric acid reagent and densitometric scanning was performed using a Camag TLC scanner III, at 420 nm. Results – The system was found to produce a compact spot for betulinic acid (R_f = 0.30). A good linear precision relationship between the concentrations (2–10 µg) and peak areas were obtained with the correlation coefficient (r) of 0.99698. The limit of detection and limit of quantification of betulinic acid were detected to be 0.4 and 2.30 µg per spot. The percentage of recovery was found to be 98.36%. The percentage relative standard deviations of intra‐day and inter‐day precisions were 0.82–0.394 and 0.85–0.341, respectively. Conclusion – This validated HPTLC method provides a new and powerful approach to estimate betulinic acid as phytomarker in the extract. Copyright © 2010 John Wiley & Sons, Ltd.     

A one-time inducible transposon for creating knockout mutants

The maize transposon Ac can move to a new location within the genome to create knockout mutants in transgenic plants. In rice, Ac transposon is very active but sometimes undergoes further transposition and leaves an empty mutated gene. Therefore, we developed a one-time transposon system by locating one end of the transposon in the intron of the Ac transposase gene, which is under the control of the inducible promoter (PR-1a). Treatment with salicylic acid induced transposition of this transposon, COYA, leading to transposase gene breakage in exons. The progeny plants inheriting the transposition events become stable knockout mutants, because no functional transposase could be yielded. The behavior of COYA was analyzed in single-copy transgenic rice plants. We determined the expression of the modified transposase gene and its ability to trigger transposition events in transgenic rice plants. The COYA element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

EXPRESSION AND PURIFICATION OF HUMAN ARP1 PROTEIN AND RAPID PREPARATION OF POLYCLONAL ANTIBODY

Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms.

Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.     

How high is the level of technical noise in microarray data?

Background  

Microarray gene expression data are commonly perceived as being extremely noisy because of many imperfections inherent in the current technology. A recent study conducted by the MicroArray Quality Control (MAQC) Consortium and published in Nature Biotechnology provides a unique opportunity to probe into the true level of technical noise in such data.     

Flow‐mediated plasticity in the expression of stickleback nesting glue genes

Nest construction is an essential component of the reproductive behavior of many species, and attributes of nests – including their location and structure – have implications for both their functional capacity as incubators for developing offspring, and their attractiveness to potential mates. To maximize reproductive success, nests must therefore be suited to local environmental conditions. Male three‐spined sticklebacks (Gasterosteus aculeatus) build nests from collected materials and use an endogenous, glue‐like multimeric protein – “spiggin” – as an adhesive. Spiggin is encoded by a multigene family, and differential expression of spiggin genes potentially allows plasticity in nest construction in response to variable environments. Here, we show that the expression of spiggin genes is affected significantly by both the flow regime experienced by a fish and its nesting status. Further, we show the effects of flow on expression patterns are gene‐specific. Nest‐building fish exhibited consistently higher expression levels of the three genes under investigation (Spg‐a, Spg‐1, and Spg‐2) than non‐nesting controls, irrespective of rearing flow treatment. Fish reared under flowing‐water conditions showed significantly increased levels of spiggin gene expression compared to those reared in still water, but this effect was far stronger for Spg‐a than for Spg‐1 or Spg‐2. The strong effect of flowing water on Spg‐a expression, even among non‐nesters, suggests that the increased production of spiggin – or of spiggin rich in the component contributed by Spg‐a – may allow more rapid and/or effective nest construction under challenging high flow conditions.     

Cloning and knockout of phytoene desaturase gene in <Emphasis Type="Italic">Sphingomonas elodea</Emphasis> ATCC 31461 for economic recovery of gellan gum

A gene encoding phytoene desaturase (crtI) in the carotenoid biosynthetic pathway of Sphingomonas elodea ATCC 31461, an industrial gellan gum-producing strain, was cloned and identified. This gene is predicted to encode a 492-amino acid protein with significant homology to the phytoene desaturase of other carotenogenic organisms. Knockout of crtI gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production by crtI gene knockout mutants is almost the same as that by the wild-type strain. In addition, a recovery method based on the colorless fermentation broth of the crtI gene knockout mutant was investigated. Compared to the volume of alcohol for the parent strain, much less alcohol (30%) is required in this recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced.     

A combination of ultrasonic-assisted extraction with RRLC-QQQ method for the determination of artemisinin in the Chinese herb Artemisia annua L

Introduction – Artemisinin, the primary active ingredient of the Chinese herb Artemisia annua L., is known to have considerable anti‐malaria properties. However, rapid, sensitive and selective method for the determination of artemisinin in it is not currently available. Objective – To develop and validate an efficient method for extraction and analysis of artemisinin from the plant samples of Artemisia annua L. by rapid resolution liquid chromatography triple quadrupole mass spectrometry (RRLC‐QQQ). Methodology – Following ultrasound‐assisted extraction (USE), RRLC‐QQQ was utilised to separate and determine artemisinin from the plant sample of Artemisia annua L. The LC separation, QQQ‐MS detection and multiple reaction monitoring (MRM) mode were optimised, and the method validation concluding selectivity, calibration, accuracy and precision, and recovery were also evaluated. Results – LC separation was performed with an isocratic elution of 20% of methanol–water (10 mmol/L ammonium acetate, pH 4.0) on a C₁₈ column. The triple quadrupole MS detection was carried out under MRM mode of precursor ion [M + H]⁺ → fragment ions m/z 265.1 and m/z 247.2. The limits of detection and quantitation of artemisinin were 0.20 and 0.75 ng/mL, respectively. The intra‐ and inter‐day precisions did not exceed 3.71%, and the deviation of the intra‐ and inter‐day mean values did not exceed ±7.50. The average recoveries for artemisinin ranged from 92.45 to 103.8% with an RSD from 2.47 to 2.79%. Conclusion – The developed RRLC‐QQQ assay is an efficient method for separation and determination of artemisinin from the plant samples of Artemisia annua L. Copyright © 2011 John Wiley & Sons, Ltd.     

Validated high-performance thin-layer chromatographic method for the quantification of thymoquinone in Nigella Sativa extracts and formulations

Introduction – The surge of interest in naturally occurring phytochemicals with anticancer potential has led to the discovery of many molecules, one of them being thymoquinone (TQ) the bioactive constituent of the volatile oil of black seed, Nigella sativa L. (NS). Objective – The aim of the present work was to develop and validate an HPTLC method for determination of TQ in NS extracts, commercially available marketed oils, polyherbal formulations and in lipid‐based oral and parenteral formulations prepared in‐house. Methodology – Analysis of TQ was performed on TLC aluminium plates pre‐coated with silica gel 60F‐254. Linear ascending development was carried out in twin trough glass chamber, saturated with mobile phase consisting of toluene–cyclohexane (8 : 2, v/v) at ambient temperature. Camag TLC scanner III was used for the spectrodensitometric scanning and analysis in absorbance mode at 254 nm. Results – The method was found to give compact spots for TQ (R_f value of 0.28 ± 0.05) and was linear over the range 100–1400 ng/spot (r² = 0.9921 ± 0.0020). Accuracy, precision and repeatability were all within the required limits. The mean recoveries measured at three concentrations were higher than 95% with RSD ≤ 3%. Conclusion – The HPTLC method developed was found to be relatively simple, rapid and accurate for the routine analysis of TQ in extracts, marketed oils, polyherbal and in‐house formulations. Copyright © 2011 John Wiley & Sons, Ltd.     

Structure characterization and identification steroidal saponins from Ophiopogon japonicus Ker-Gawler (Liliaceae) by high-performance liquid chromatography with ion trap mass spectrometry

Introduction – Steroidal saponins are the main active constituents in Ophiopogon japonicus Ker‐Gawler (Liliaceae). However, because of their high polarity, non‐chromophores and low content in plants, steroidal saponins are difficult to be isolated from O. japonicus by conventional phytochemical methods. Objective – To develop a sensitive and rapid approach towards the structural analysis of steroidal saponins using HPLC/ESI‐MSⁿ. Methodology – The fragmentation behaviors of six known steroidal saponins in negative ESI‐MSⁿ were used to deduce their mass spectral fragmentation mechanisms. By using HPLC/ESI‐MSⁿ, the important structural information on aglycone types, sugar types and saccharide sequences can be obtained. Results – According to the HPLC retention behaviour, the molecular structural characteristics provided by multistage mass spectrometry spectra and the literature, a total of 8 steroidal saponins were tentatively identified or characterized in O. japonicus rapidly. Conclusion – This work has shown that HPLC‐ESI‐MSⁿ may be used as an effective and rapid method for the characterization and identification of steroidal saponins from O. japonicus. Copyright © 2010 John Wiley & Sons, Ltd.     

乙醛脱氢酶基因ALDH2对雄性小鼠生育能力的影响

本研究使用乙醛脱氢酶2(ALDH2)基因全敲除的雄性C57BL/6(B6)小鼠（Musmusculus domesticus）,通过分析不同周龄小鼠的睾丸脏器系数、睾丸组织细胞形态、精子运动参数、配种后母鼠产仔数及子代雄雌比等生育指标,探讨ALDH2基因敲除对雄性小鼠生育能力的影响。结果表明,与野生型（WT）小鼠相比,5、7、10周龄ALDH2基因敲除型（KO）雄性小鼠睾丸脏器系数显著降低（P <0.05）;睾丸组织细胞间质变大,精子活率显著降低（P <0.05）;产仔数和雄雌比显著降低（P <0.05）。本研究为揭示乙醛脱氢酶ALDH2基因在雄性小鼠生殖中的作用提供了一定的基础。     

Purification, cloning, and characterization of an arylsulfotransferase from the anaerobic bacterium Eubacterium rectale IIIH

A bacterium, Eubacterium rectale IIIH, which possessed arylsulfotransferase (ASST) activity was isolated from human feces. The ASST gene (astA) was cloned and the corresponding protein partially characterized. This gene shows only moderate homology to the previously sequenced ASST genes of Klebsiella and Enterobacter, which are very closely related to each other. Journal of Industrial Microbiology & Biotechnology (2000) 25, 305–309. Received 02 August 2000/ Accepted in revised form 19 November 2000     

Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases

Summary: Targeted mutagenesis of genes‐of‐interest, or gene‐knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN‐mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN‐mediated germ cell mutagenesis. genesis 52:431–439, 2014. © 2014 Wiley Periodicals, Inc.